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Il protocollo segue le linee guida del comitato etico di University Hospital Zurich umana ricerca.
1. diagnosi del Neuromata e selezione del paziente
<ol><li>Eseguire preoperatori blocchetti di nervo prossimali al neuroma al fine di confermare la diagnosi. Valutare i blocchi nel terzo centrale dell'avambraccio ed escludere potenziali inneschi delle aree sovrapposte di innervazione di SBRN, come il nervo cutaneo antebrachial laterale (LACN), nervo interosseous posteriore (PIN) e un ramo dorsale del nervo ulnare 10.</li>
	<li>Eseguire un intervento chirurgico solo in pazienti con dolore notevole riduzione (almeno 5 punti su scala analogica visiva (VAS) per il dolore)11.</li>
</ol>2. preparazione chirurgica
<ol><li>Proibire il paziente qualsiasi assunzione di cibo o liquido 6 h prima della chirurgia.</li>
	<li>Collocare il paziente al centro la suite chirurgica su un tavolo operatorio universale in una posizione di sedia di spiaggia.</li>
	<li>Posizionare il braccio/mano commovente su un bracciolo.</li>
	<li>Chiedi l'anestesisti per eseguire anestesia endotracheale (fentanyl 2 µ g/kg, rocuronio 0,6 mg/kg, propofol 150-250 mg).</li>
	<li>Disinfettare la pelle con povidone iodio nella zona della liposuzione (ad es. interno coscia e/o dell'addome superiore ed inferiore) e il sito dell'asportazione neuroma.</li>
	<li>Coprire il paziente con teli chirurgici sterili, lasciando esposto il sito della liposuzione e del sito chirurgico.</li>
</ol>3. tumescente liposuzione
<ol><li>Fare diverse incisioni piccola pugnalata (puntura) di circa 3-5 mm di diametro con un bisturi n. 11-lama nella zona di liposuzione. Incise per quanto riguarda lo strato sottocutaneo è raggiunto.</li>
	<li>Iniettare la soluzione tumescente contenente un diluito lidocaina ed epinefrina (soluzione di lattato di Ringer 500 mL, 20 mL di lidocaina 1% e di epinefrina 1mg) tramite l'infiltrator 14 gauge (cannula di infiltrazione) nello strato di grasso sottocutaneo (3 mm di profondità) nella regione di raccolta grasso.</li>
	<li>Attendere almeno 20 min consentendo la soluzione tumescente effettive.</li>
	<li>Gancio 3 mm-raccolta cannula (diametro esterno 3 mm, lunghezza 26 cm) ad una siringa da 60 mL Toomey e inizio raccolta adiposo tramite le incisioni lo stab. Nota: La mano non dominante controlla continuamente posizionamento e corso della cannula.</li>
	<li>Tirare indietro lo stantuffo della siringa Toomey per il marchio di 60 mL e posizionare un morsetto di zanzara lungo lo stantuffo ritirato al fine di mantenere una pressione negativa nella siringa e una continua creazione di vuoto per evitare la mano dominante.</li>
	<li>Raccogliere un minimo di 220 mL di tessuto adiposo posizionando le siringhe Toomey in posizione verticale nel basamento della siringa Toomey fornito; Questo permette la sedimentazione del grasso e la separazione dal liquido.</li>
	<li>Prestare particolare attenzione per ottenere un innesto grasso di buona qualità senza notevoli quantità di sangue. Nota: Cambiare la regione di liposuzione quando aumentata sangue contenuto è notato.</li>
	<li>Le incisioni di pugnalata con semplici punti di sutura e applicare una pressione come richiesto (ad es. compressione cintura).</li>
	<li>Trasferire il lipoaspirato in un set monouso sterile monouso del sistema di isolamento.</li>
	<li>Tenere una siringa Toomey con lipoaspirato nello stand fornito al fine di ottenere la frazione grassa per successive lipofilling.</li>
</ol>4. tessuto adiposo elaborazione
<ol><li>Chiudere il set di consumabili sterile. Nota: Il sistema di isolamento (Vedi Tabella materiali) conferma la completezza eseguendo un test bagnato.</li>
	<li>Seguire le istruzioni sul display. Nota: Il processo è per lo più software controllato e richiede solo la conferma manuale per i passaggi elencati di seguito, premendo i pulsanti "Avanti" o "indietro" sul pannello di controllo. Il lipoaspirato viene trasferito nel contenitore di raccolta del tessuto. Nel recipiente di raccolta del tessuto, il lipoaspirato è pesato e automaticamente pulito con soluzione di Ringer lattato al fine di rimuovere le cellule residue di sangue e la soluzione bagnante.</li>
	<li>Se necessario, aggiungere più grasso. Nota: A seconda del peso del tessuto adiposo raccolto il sistema di isolamento chiede all'utente di aggiungere più grasso o si calcola la quantità di reagente enzimatico aggiuntive da aggiungere.</li>
	<li>Aggiungere la quantità necessaria di reagente enzimatico quando richiesto dal sistema di isolamento. Nota: Durante il processo di digestione enzimatica, si ottiene la separazione del lipido e la SVF. In tal modo, il tessuto è in movimento oscillante. Una volta completata la digestione enzimatica, la SVF viene trasferito automaticamente nella camera della centrifuga per la concentrazione delle cellule. Camera della centrifuga, la SVF è concentrata per un pellet cellulare (velocità di centrifuga è circa 3.000 x g). Infine, il pellet cellulare subisce una serie di lavaggi con soluzione di Ringer lattato per ridurre i livelli dell'enzima residua nell'output. Quando il processo è completato, si ottiene 5 mL di liquido chiaro (SVF).</li>
</ol>5. intervento chirurgico
<ol><li>Per approcciare il neuroma, fare un'incisione della pelle sopra il sito di neuroma precedentemente diagnosticato nel terzo distale dell'avambraccio.</li>
	<li>Sotto l'uso di bicchieri di ingrandimento chirurgico occhialini binoculari (2.5-3.5 X), continuare a sezionare il tessuto sottocutaneo e identificare il nervo implicato prossimale al neuroma.<ol>
<li>Successivamente, segui il nervo distale e identificare il neuroma (basato su ispezione visiva).</li>
	</ol>
</li>
	<li>Una volta che l'estensione completa del neuroma è chiaro, iniziare asportare il neuroma da un taglio dritto trans-neurale scissor. Se nessun neuroma viene identificato, viene eseguita solo una neurolisi del nervo.</li>
	<li>Continuare con il neurolysis del moncone del nervo rimanenti fino a 3 cm prossimale al polso. Nota: L'estremità del moncone del nervo deve essere situato nel centro dell'incisione principale.</li>
	<li>Organizzare due-quattro piccola puntura (pugnalata) incisioni (1 mm di diametro) con un bisturi attraverso epidermico e dermico cutis intorno l'approccio principale.</li>
	<li>Posizionare due o quattro cannule smussati (16-gauge, lunghezza 9 cm) tramite la pugnalata incisioni perineural al ceppo. Nota: La punta delle cannule deve essere indirizzata verso il moncone del nervo.</li>
	<li>Fissare le cannule al posto di medicazione adesiva.</li>
	<li>Posizionare i punti di sutura intorno le cannule e lasciare il materiale di sutura in situ senza fissarne il nodo.</li>
	<li>Sutura l'incisione principale strettamente al fine di evitare il deflusso di innesto. Nota: Assicurarsi che le cannule rimangono in situ.</li>
</ol>6. applicazione dell'innesto grasso SVF-arricchita
Nota: Dopo circa 1,5 h di elaborazione, i 5 mL della SVF trasformati è pronto per un ulteriore uso.
<ol><li>Aspirare 5 mL di SVF in siringa 10 mL comunicante.</li>
	<li>Allo stesso modo, aspirare 2 mL della frazione lipidica sedimentato nella siringa 10 mL avversaria.</li>
	<li>Mescolare i 5 mL di SVF con 2ml della frazione lipidica sedimentato alternando tirando indietro lo stantuffo di una siringa e tirando giù lo stantuffo della siringa comunicante, avversaria fino a quando si ottiene una miscela sufficiente.</li>
	<li>Dividere l'innesto grasso SVF-arricchita ugualmente in due-quattro siringhe da 10 mL.</li>
	<li>Distribuire l'innesto grasso SVF-arricchita preparato uno dopo l'altro ugualmente intorno al nervo ceppo collegando le siringhe per le cannule smussate. Nota: Prima di applicare l'innesto, assicurarsi che le cannule sono fissate nella posizione corretta e fare attenzione a non slogarvi le cannule durante l'applicazione di innesto grasso.</li>
	<li>Rimuovere con cautela le cannule.</li>
	<li>Poi, il nastro le incisioni di piccola puntura con steri-strips.</li>
	<li>Immobilizzare la mano applicando una stecca nell'intrinseca più posizione (giunti metacarpophalangeal (MCP) flesso a 60-70°, articolazioni interfalangee (IP) completamente estesi, pollice nella proiezione pugno, polso in flessione dorsale di 25°). Se solo viene eseguita neurolisi del nervo, non è necessaria nessuna stecca e braccio e del polso sono bendati per 10 giorni.</li>
</ol>7. postoperatorio movimentazione dei pazienti
<ol><li>Trasferire il paziente per la post-chirurgia unità di recupero post-anestesia e consentono un monitoraggio sufficiente del rione.</li>
	<li>Continuare immobilizzare il polso per 10 giorni dopo l'intervento chirurgico.</li>
	<li>Eseguire il primo cambio di bende dopo post-chirurgia 48h e continuare ad intervalli regolari (cioè ogni secondo giorno fino alla dimissione).</li>
	<li>Rimuovere le suture 12-14 giorni postoperatorio.</li>
	<li>Eseguire la valutazione clinica del dolore durante il follow-up a 2, 6, 12 e 36 mesi.</li>
</ol>

<ol>
	<li>Mackinnon, S. E., Dellon, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+treatment+of+recurrent+dorsoradial+wrist+neuromas.">Results of treatment of recurrent dorsoradial wrist neuromas.</a> Ann Plast Surg. 19 (1), 54-61 (1987).</li><li>Hazari, A., Elliot, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+end-neuromas,+neuromas-in-continuity+and+scarred+nerves+of+the+digits+by+proximal+relocation.">Treatment of end-neuromas, neuromas-in-continuity and scarred nerves of the digits by proximal relocation.</a> J Hand Surg Br. 29 (4), 338-350 (2004).</li><li>Goldstein, S. A., Sturim, H. 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Lo scopo del presente studio è quello di metodicamente illustrano gli aspetti tecnici del SVF-arricchito l'innesto grasso come un nuovo trattamento per il fine-neuromata sintomatico.
Neuromata della SBRN è associati con un tasso di successo chirurgico di solo 33%12. Vaienti et al ha presentato una serie di risultati favorevoli in follow-up iniziale utilizzando innesto grasso perineural (PFG) senza aggiungere la SVF in 8 pazienti con neuromata...

La gestione del fine-neuromata sintomatico del SBRN rimane impegnativa con una moltitudine di trattamenti chirurgici esistenti. Principalmente, il neuroma è asportato e il restante moncone del nervo viene trasferito in strutture vicine quali muscoli1,2, ossa3o vene4. Questo è presupposto per evitare il fuori-germoglio di fasci neurali del rigeneranti imitando l'atmosfera di un fine-organo. Tuttavia, nessuna di queste tecniche ha provato il superiore, come spesso sono stati osservati elevati tassi di recidiva di dolore e riduzione del dolore insufficiente.
Recentemente, l'innesto di grasso attorno al moncone del nervo rimanenti dato risultati promettenti e fu sostenuto come una nuova opzione di trattamento per fine-neuromata dolorosa dell' arto superiore5. Oltre ad agire come barriera meccanica per impedire il movimento continuo e toccare il dolore, l'aspetto benefico di innesto di grasso è piuttosto attribuita a livello cellulare. Tessuto adiposo umano contiene diversi tipi di cellule, comprese preadipociti, mastociti, periciti, cellule endoteliali, cellule muscolari lisce, fibroblasti, e adiposo derivate cellule staminali (CSTA) insieme con le cellule staminali emopoietiche6. Questa popolazione cellulare eterogenea è indicata come Adipose-Derived rigenerazione cellule (ADRC) o SVF e può essere ottenuta dal lipoaspirato da un sistema di isolamento (Vedi La tabella materiali) e reagente enzimatico applicata (Vedi tabella materiali )7. L'esatto meccanismo d'azione è ancora non pienamente compreso; Tuttavia, SVF è stato associato con i miglioramenti nella angiogenesi, immunomodulazione, differenziazione e secrezione della matrice extracellulare8.
La popolazione delle cellule concentrato della SVF viene aggiunto all'innesto grasso con lo scopo di ridurre il tasso di riassorbimento e di aumentare il potenziale rigenerativo dell'innesto grasso, come gli studi recenti hanno rivelato dolore ricaduta dopo 12 mesi con l'innesto grasso senza aggiunto SVF 5. Inoltre, l'azione biologica fornisce anche effetti benefici sul nervo stesso riducendo germogliare axonal disorganizzato di ceppo9 e aderenze successive cicatrice che potrebbero provocare l'allettamento del nervo.
Il primo studio clinico per descrivere SVF-arricchito l'innesto grasso come una romanzo opzione chirurgica per il trattamento di fine-neuromata sintomatico di SBRN è stato recentemente pubblicato10. In generale, la tecnica può essere applicata qualsiasi neuroma che accade dopo la ferita del nervo accidentali o iatrogeni. L'obiettivo del presente studio successivo è metodicamente illustrare gli aspetti tecnici di questa procedura.

<table border="0" cellpadding="0" cellspacing="0" width="688">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Cytori Celution System</td>
			<td style="width:71px;">Cytori</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">isolation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Celase</td>
			<td style="width:71px;">Cytori</td>
			<td style="width:119px;"></td>
			<td>enyzme reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">liposuction system</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">tumescent solution (500 ml Lactated Ringers, 20 ml of 1% lidocaine and 1 mg epinephrine)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14-gauge infiltrator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mm-collecting cannula (15, 26, 32 cm)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mL Tommey syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mosquito clamp</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toomey syringe stand</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">4 cannulas (fat injecting cannulas 1.5 mm, syringe NO.2, 90 mm)</td>
			<td style="width:71px;">Chalybs</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">10 mL syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">connecting device for syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">#11- and #15-blade scalpel</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">stevens scissors</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">pins pincette</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">bipolar electrocautery</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">suture material</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">adhesive dressing</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">binocular loupes magnifier glasses (2.5-3.5x)</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">Statistical Package for Social Sciences, version 22</td>
			<td>IBM</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

stromal vascular fraction-enriched fat grafting for the treatment of symptomatic end-neuromata

Lo scopo di questo studio era di metodicamente illustrare ed evidenziare i passaggi cruciali della frazione vascolare stromal (SVF)-arricchito l'innesto grasso come un trattamento del romanzo di fine-neuromata sintomatico dei nervi sensoriali periferici e in questo studio, in particolare di il ramo superficiale del nervo radiale (SBRN). Nonostante una moltitudine di trattamenti esistenti, dolore postoperatorio persistente e comune dolore ricaduta sono ancora molto comune, indipendente della procedura valutata. Il neuroma è asportata microsurgically protocollo di conseguenza a standardizzato. Invece la rilocazione del moncone del nervo rigenerante nelle vicine strutture anatomiche, come il muscolo o osso, un innesto grasso è applicato perifocally e funge da barriera meccanica. Al fine di ridurre il tasso di riassorbimento del grasso e aumentare il potenziale rigenerativo dell'innesto, la SVF altamente concentrato è integrato nell'innesto. La SVF è isolato dal grasso sottocutaneo da separazione enzimatica e meccanico di lipoaspirato da un sistema di isolamento commerciale specifico. L'innesto di grasso SVF-arricchito fornisce sia una barriera meccanica e vari effetti biologici a livello cellulare, compreso il miglioramento l'angiogenesi, l'infiammazione e la fibrosi. Effetti sia meccanici che biologici aiutano a ridurre l'escrescenza disorganizzato axonal del moncone del nervo durante la rigenerazione del nervo e quindi evitare il ripetersi di doloroso fine-neuromata.

I dati sono stati analizzati utilizzando il software statistico e presentati come significa ± deviazioni standard. Il test di Kolmogorov-Smirnov è stato applicato per verificare la distribuzione non parametrica della popolazione dello studio e confermato una distribuzione empirica negativa. Basato sulla natura della popolazione dello studio non parametrica, test di Wilcoxon per gli esempi correlati è stato impiegato per confrontare i risultati preoperative e postoperatori. Significativ...

Watch this Scientific Journal Video about Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata at JoVE.com

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

Stromal arricchito in frazione grassa l'innesto vascolare per il trattamento di fine-neuromata sintomatica

Lo scopo dello studio è quello di illustrare il procedimento tecnico della frazione vascolare stromal (SVF)-arricchito l'innesto grasso come un trattamento del romanzo di fine-neuromata sintomatico. Questa tecnica offre vantaggi della barriera meccanica e l'azione biologica a livello cellulare della SVF trasformati e concentrato.

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

stromal-vascular-fraction-enriched-fat-grafting-for-treatment

Research

JoVE Journal

Medicine

7.6K Views.  University Hospital Zurich. Lo scopo dello studio è quello di illustrare il procedimento tecnico della frazione vascolare stromal (SVF)-arricchito l'innesto grasso come un trattamento del romanzo di fine-neuromata sintomatico. Questa tecnica offre vantaggi della barriera meccanica e l'azione biologica a livello cellulare della SVF trasformati e concentrato.

Video: Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Generazione sperimentale di carcinoma-Associated fibroblasti (CAF) da fibroblasti umani mammari

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Ricerca

Medicina

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Aortica trapianto ortotopico in Mouse per lo studio della malattia vascolare

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Aspetti tecnici della fistola aorto-cavale mouse

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tridimensionale Co-cultura Modello di tumore stromale-Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Trapianto nel Mouse Pad ovarico Fat

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Network Analysis di forame ovale elettrodi Recordings in farmaco-resistenti epilessia del lobo temporale pazienti

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

Veicolazione Trans-timpanico per il trattamento dell'ototossicità

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Protocollo clinico di produrre tessuto adiposo-derivato Stromal frazione vascolare per potenziale rigenerazione della cartilagine

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Isolamento di cellule rigenerative di derivazione adiposa per il trattamento della disfunzione erettile a seguito di prostatectomia radicale

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Istituzione di un modello suina di infarto post-miocardico per il trattamento delle cellule staminali

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...