This work was supported by an Australian Research Council Grant DP110100642 (to PT) and National Health and Medical Research Council Grants APP1002520 and APP1059426 (to PT and HYG).

NOTA: Tutte le presenti esperimenti sono stati eseguiti secondo le locali procedure di etica animale dell&#39;Università di Queensland (approvati dalla University of Queensland, anatomico Biosciences Comitato Etico). 1. Islet Isolamento <ol><li> Preparazione dei reagenti <ol><li> Miscela di enzimi <ol><li> Per la digestione pancreatica, utilizzare una miscela di liberase e collagenasi tipo IV. Diluire liberase TL (termolisina basso) 1 flacone di 5 mg con 26 ml di DMEM (Dulbecco Modified Eagle Medium). </li><li> Preparare collagenasi di tipo IV in tampone di Hank ad una concentrazione di 0,5 mg / ml, supplementato con 5 mM HEPES, 0.5mm CaCl 2, 0,1 mg / ml e DNAsi 1mg / ml sieroalbumina bovina. </li><li> Mescolare il TL liberase e soluzioni collagenasi in un rapporto di 4: 1 (in volume), cioè, 4 ml liberase TL + 1 ml di collagenasi. Aliquota della miscela di enzimi per 2,5 ml ciascuna e conservare a -20 ° C. NOTA: Le ore di incubazione tessuto vary un po &#39;tra i lotti di enzima (30 sec a differenza di 1 min). Pertanto, un test di gruppo può essere richiesto. </li></ol></li><li> RPMI Isolamento media <ol><li> Sciogliere una fiala di RPMI 1640 polvere in 1 l di acqua a temperatura ambiente e integrare con 2 g NaHCO 3, 4.02 g HEPES. </li><li> Combina i media isolamento RPMI in una grande beuta, mescolare con agitatore magnetico, regolare a pH 7,4 con NaOH, filtro sterilizzare e conservare a 4 ° C. </li></ol></li><li> RPMI Media Cultura </li><li> Utilizzare coltura RPMI con 10% di siero fetale bovino e 1% di antibiotici (ad esempio, penicillina 100 U / ml, streptomicina 100 mg / ml). </li></ol></li><li> Isolamento Islet e Coltura <ol><li> Sacrificio animale <ol><li> Sacrificio topi di dislocazione cervicale o di CO 2 asfissia secondo le procedure di etica dell&#39;istituzione locale. Spruzzare accuratamente il corpo del mouse con il 70% di etanolo. </li></ol></li><li> Perfusione pancreas <ol><li> USE 2 tagli lunghi lateralmente attraverso la pelle addominale e del peritoneo per fare un lembo a forma di V. Tirare il lembo della pelle fino a scoprire l&#39;intera cavità addominale, messo da parte dell&#39;intestino e mantenere il fegato in posizione da una carta velina ripiegata per rivelare il dotto biliare comune (figure 1 e 2). Mettere l&#39;animale in modo che la testa verso il chirurgo e la coda dal chirurgo. </li><li> Applicare un morsetto vascolare nella ampolla sulla parete del duodeno per bloccare gli enzimi iniettati di entrare duodeno (Figura 1). NOTA: Questo è un passo importante in quanto determina se gli enzimi iniettati andranno nel dotto pancreatico e profumato il pancreas o reflusso al sito di iniezione (oltre morsetto) o andare nel duodeno (sotto pinza). </li><li> Incannulare dotto biliare comune con un ago G 31 nei pressi dello svincolo del dotto epatico comune e dotto cistico (Figura 1) e lasciare spazio per una seconda iniezione se the pancreas non riesce a perfusione la prima volta. Lentamente (sopra ~ 10 sec) iniettare circa 2 ml di miscela di enzimi freddo nel mouse dotto biliare comune. NOTA: Il pancreas saranno perfusi rapidamente se la pinza è nella posizione corretta. </li><li> Regolare la posizione di blocco se l&#39;ago si trova all&#39;interno del dotto biliare comune, ma non può profumato pancreas (es., Spostare la pinza un po &#39;lontano dal fegato se oltre bloccato o spostarlo verso il basso un po&#39; per il fegato se sotto bloccato). NOTA: L&#39;ago volte va nella capsula circostante piuttosto che il lume del dotto. Un segno utile per verificare se l&#39;ago è all&#39;interno del dotto biliare comune è la dilatazione del condotto d&#39;iniezione. Se il pancreas non riesce a perfusione, un&#39;alternativa è la miscela enzima potrebbe essere iniettato a più siti del pancreas con minore rendimento previsto. La perfusione del sito sinistra del pancreas (lobo splenica) è importante per la massimizzazione della resa isolotto. </li><li> Dopo il pancreas è perfused, rimuovere rapidamente il pancreas, metterla in una fiala (~ 20 ml, vetro) e incubare per ~ 19 min in un bagno di 37 ° C Acque. NOTA: Il tempo di incubazione può variare un po &#39;a seconda del ceppo di topo ed età. </li><li> Dopo il tempo di incubazione, arrestare il processo di digestione aggiungendo ~ 20 ml di terreni di isolamento freddo. Agitare delicatamente il flacone di interrompere il pancreas e versare attraverso un normale colino del tè (diametro dei pori di 1 mm) in una sterile tubo da 50 ml. Rabboccare il tubo con terreni di isolamento e centrifugare a 400 xg per 1 min a 4 ° C con freno off. </li><li> Sciogliere di nuovo il pellet in terreni di isolamento e trasferire due provette da 15 ml, quindi si centrifuga a 400 xg per 1 min a 4 ° C con freno off. Dopo l&#39;aspirazione del surnatante, miscelare i due palline bene con 6 ml di mezzi di separazione cellulare gradiente di densità (Histopaque soluzione 1077) per provetta nel vortex o pipettando su e giù. Poi aggiungere delicatamente ~ 6 ml dei terreni di isolamento al lato del tubo in cimai mezzi di separazione gradiente di densità e centrifugare a 100 xg per 15 min a 4 ° C con freno off. </li><li> Raccogliere il surnatante nella parte superiore (terreni di isolamento), media e bassa (densità cellulare gradiente di mezzi di separazione) strato in 3 piatti separati. Handpick isolotti in questi 3 piatti, usando una pipetta sotto un microscopio da dissezione. NOTA: Lo strato intermedio contiene la maggior parte degli isolotti. Isolotti sono riconosciuti come strutture compatte di ~ 50 a 500 micron di dimensione (Figura 3). Otteniamo ~ 200 isolotti in topi WT e 300- &lt;100 isolotti nei topi db / db. </li></ol></li><li> Islet Culturing <ol><li> Per aiutare gli isolotti riprendersi dalla digestione enzimatica, gruppi culturali di 40 isolotti in un 25 centimetri capsula di Petri con 6 ml RPMI terreni di coltura (10mM glucosio) a 37 ° C, 5% / 95% di CO 2 / O 2 per 2 giorni prima dal vivo -cell 2-photon imaging. Modificare il terreno di coltura quotidiano. </li></ol></li></ol></li></ol> 2. Live Cell 2-Photon Imaging <ol><li> Preparazione dei reagenti <ol><li> Extracellulare Buffer <ol><li> Preparare il sodio ricco tampone extracellulare per il test 2-fotone con 140 mM NaCl, KCl 5 mM, 2,5 mM CaCl 2, 1 mM MgCl 2, 5 NaHCO 3 e 10 mM HEPES mM. </li></ol></li><li> Extracellulare Dye <ol><li> Per il saggio 2 fotoni, preparare magazzino SRB 8 mm in tampone extracellulare, aliquota in piccoli tubi e conservare a -20 ° C. Diluire lo stock SRB 1:10 in tampone fresco extracellulare per ottenere una concentrazione di lavoro di 0,8 mm. </li></ol></li></ol></li><li> fotone Imaging <ol><li> Innanzitutto, pre-incubare 2 giorni isolotti coltivate in 3 mM glucosio tampone extracellulare per 30 min. NOTA BENE: Usiamo isolotti tra 2 e 5 giorni in coltura. </li><li> Quindi, inserire un vetrino sulla camera termo-controllo montato sul palco microscopio. Utilizzare del grasso per impedire la fuoriuscita di soluzione. </li><li> Coprire la camera con 500 ml di ExtracellBuffer colare contenente 0,8 SRB mm con differenti concentrazioni di glucosio (3 mm, 6 mm, 15 mm e 20 mm) di glucosio. </li><li> Quindi, posizionare l&#39;isolotto di pre-incubazione nel mezzo della camera e si concentrano a 3-4 strati di cellule nel isolotto dove, nelle isole di roditori, immunocolorazione per l&#39;insulina mostra che la maggior parte delle cellule sono cellule beta. NOTA: Il SRB illustrerà tutte le membrane delle cellule dell&#39;isolotto rapidamente e l&#39;isolotto è pronto per l&#39;immagine. </li><li> Utilizzare un microscopio 2 fotoni con un obiettivo 60X immersione in olio (NA 1.42) e software di imaging (ad es., Scanimage 11). La Figura 4 illustra una tipica disposizione microscopia. Immagine eventi esocitosi utilizzando SRB come una membrana impermeant marcatore extracellulare fluorescente eccitato da impulsi laser a femtosecondi a 880 nm, con la fluorescenza rilevati a 550-650 nm. Registrare le risposte isolotto alla stimolazione. </li><li> Identificare gli eventi esocitosi singoli dall&#39;improvvisa apparizione di una piccola macchia fluorescente (risoluzione 10 pixel /1; m) e confermare con la fase veloce di salita del segnale fluorescente sopra una regione di interesse (~ 0,78 micron 2). </li></ol></li></ol>

<ol>
	<li>Guariguata, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035.">Global estimates of diabetes prevalence for 2013 and projections for 2035.</a> Diabetes Research and Clinical Practice. 103, 137-149 (2014).</li><li>Ashcroft, F. M., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus+and+the+beta+cell:+The+last+ten+years.">Diabetes mellitus and the beta cell: The last ten years.</a> Cell. 148, 1160-1171 (2012).</li><li>Weir, G. C., Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+stages+of+evolving+beta-cell+dysfunction+during+progression+to+diabetes.">Five stages of evolving beta-cell dysfunction during progression to diabetes.</a> Diabetes. 53, S16-S21 (2004).</li><li>Kjorholt, C., Akerfeldt, M. C., Biden, T. J., Laybutt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hyperglycemia,+independent+of+plasma+lipid+levels,+is+sufficient+for+the+loss+of+beta-cell+differentiation+and+secretory+function+in+the+db/db+mouse+model+of+diabetes.">Chronic hyperglycemia, independent of plasma lipid levels, is sufficient for the loss of beta-cell differentiation and secretory function in the db/db mouse model of diabetes.</a> Diabetes. 54, 2755-2763 (2005).</li><li>Wang, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+type+2+diabetic+rodent+models.">Spontaneous type 2 diabetic rodent models.</a> Journal of Diabetes Research. , (2013).</li><li>Hummel, K. P., Dickie, M. M., Coleman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+a+new+mutation+in+mouse.">Diabetes a new mutation in mouse.</a> Science. 153, 1127-1128 (1966).</li><li>Do, O. H., Low, J. T., Gaisano, H. Y., Thorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretory+deficit+in+islets+from+db/db+mice+is+mainly+due+to+a+loss+of+responding+beta+cells.">The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells.</a> Diabetologia. 57, 1400-1409 (2014).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+principally+regulates+insulin+secretion+in+mouse+islets+by+controlling+the+numbers+of+granule+fusion+events+per+cell.">Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell.</a> Diabetologia. 56, 2629-2637 (2013).</li><li>Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet.">Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet.</a> Science. 297, 1349-1352 (2002).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion+from+beta+cells+in+intact+mouse+islets+is+targeted+towards+the+vasculature.">Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature.</a> Diabetologia. 57, 1655-1663 (2014).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomedical Engineering Online. 2, 13 (2003).</li></ol>

The authors declare they have no competing financial interests.

Il fattore più critico nell&#39;isolamento isolotto è la perfusione iniziale del pancreas; un sotto perfuso pancreas risultati in una resa isolotto notevolmente inferiore. Altri fattori influenzano anche la qualità di isolamento, come il tempo di digestione e il livello di agitazione che potrebbe in parte compensare il livello di perfusione. Ad esempio, un pancreas pienamente perfuso dovrà essere incubate a 37 ° C per ~ 18 min 30 sec agitatore, al contrario un sotto digerito pancreas possono richiedere ~ 20 min dig...

Il ruolo della disfunzione delle cellule beta nella malattia è stato ampiamente riconosciuto 3,4. Le linee cellulari, come il MIN6 e INS-1 sono strumenti utili per comprendere la biologia del comportamento delle cellule beta. Tuttavia, il controllo fisiologico della secrezione di insulina avviene all&#39;interno delle isole di Langerhans. Questi isolotti contengono migliaia di cellule beta fitte, così come i vasi sanguigni e di altri tipi di cellule endocrine. Questo ambiente all&#39;interno dell&#39;isolotto influenza la secrezione di insulina, ed è probabile che sia importante nel diabete. Pertanto, per comprendere il controllo fisiologico della secrezione di insulina, e la fisiopatologia della malattia, è essenziale studiare isolotti intatti. Isolamento Islet Isole umane vive e, in particolare, isolotti umani provenienti da pazienti affetti da diabete di tipo 2 sono difficili da ottenere. Inoltre, isole umane hanno limitate possibilità di manipolazione molecolare sperimentale. I ricercatori hanno quindi impiegato èlascia da animali e modelli animali di diabete di tipo 2. Un tale modello di malattia è il mouse db / db. Si tratta di una mutazione spontanea che i modelli di diabete di tipo 2 con una progressione fenotipo che ricorda molto da vicino la malattia umana 5,6. Il protocollo presentato qui per l&#39;isolamento delle isole da diabetici db / db topo ha molti punti in comune con gli altri gruppi con alcuni filtri per migliorare la resa, la purificazione e la sopravvivenza delle isole migliorata. Imaging 2 fotone Il live-cella 2 fotoni test qui descritta permette ai ricercatori di quantificare il numero e valutare le caratteristiche dei singoli granuli insulina contenente da molte cellule del diabetico 7 e wild type isolotti 8,9.

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liberase TL 5mg</td>
			<td style="width:173px;">Roche</td>
			<td style="width:119px;">-5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase type IV-1g</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histopaque 1077 500ml</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium (10X) 1L</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">R1383</td>
			<td>to prepare isolation media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 (1X) 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">21870-076</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin 100ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>to prepare cultured media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">10099-141</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">11966-025</td>
			<td>to dilute the liberase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metamorph program</td>
			<td style="width:173px;">Molecular Devices, USA</td>
			<td style="width:119px;"></td>
			<td>to analyze the 2-photon images</td>
		</tr>
	</tbody>
</table>

leprdb mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets

Il diabete di tipo 2 è una malattia cronica che colpisce 382 milioni di persone nel 2013, e si prevede un aumento di 592 milioni entro il 2035 1. Nel corso degli ultimi 2 decenni, il ruolo di una disfunzione delle cellule beta nel diabete di tipo 2 è stato chiaramente stabilito 2. Progresso della ricerca ha richiesto metodi per l&#39;isolamento di isole pancreatiche. Il protocollo di isolamento delle isole qui presentata condivide molte fasi comuni con protocolli di altri gruppi, con alcune modifiche per migliorare la resa e la qualità delle isole isolate sia dal tipo selvatico e diabetico db LEPR (db / db) topi. Un live-cell imaging metodo 2-fotone è quindi presentata che può essere utilizzato per studiare il controllo della secrezione insulinica entro isolotti.

Resa Islet e purificazione Per un normale topo wild-type, sono attesi circa 200 isolotti. Isolotti sani guardare luminoso, di forma circolare e un bordo liscio. Un lotto di isolamento su digerito di solito ha piccole e sfocate isolotti mentre un partita sotto-digerito ha meno isolette e isolotti di cellule attaccato acinar (Figura 3). Per diabetici db / db topo, la resa isolotto e l&#39;aspetto dipende dalla progressione della malattia con migliori topi glicemico sono più g...

Watch this Scientific Journal Video about Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets at JoVE.com

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

LEPR<sup&gt; Db</sup&gt; Mouse Modello di diabete di tipo 2: pancreas isolamento Islet e live-cella 2-Photon Imaging di isolotti Intact

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

leprdb-mouse-model-type-2-diabetes-pancreatic-islet-isolation-live

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

9.4K Views.  The University of Queensland. We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Video: Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Isolamento di isole umane da pazienti parzialmente Pancreatectomized

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Ricerca

Medicina

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocolli di colorazione di isole pancreatiche umane

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Quantificare permeabilità glomerulare delle Macromolecole fluorescenti usando la microscopia a due fotoni a Monaco di Baviera ratti Wistar

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocollo migliorato per microdissezione laser di isole pancreatiche umane da campioni chirurgici

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

Isolamento, Cultura e Imaging di Human Fetal pancreatici gruppi di cellule

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Contrasto dinamico migliorato risonanza magnetica di un modello ortotopico cancro del pancreas mouse

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Lesioni chirurgica al pancreas mouse attraverso la legatura del Dotto pancreatico come un modello per Endocrine e Esocrine riprogrammazione e la proliferazione

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal trapianto di isole pancreatiche nel modello del topo

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Longitudinale in Vivo Imaging e quantificazione dell'isolotto pancreatico umano Innesto e contribuendo cellule ospitanti nella camera occhio anteriore

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

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