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).

HINWEIS: Alle vorliegenden Experimenten wurden nach den örtlichen Tierethik Verfahren der University of Queensland (von der University of Queensland, Anatomisches Biosciences Ethikkommission genehmigt) durchgeführt. 1. Islet Isolation <ol><li> Vorbereitung der Reagenzien <ol><li> Enzymgemisch, <ol><li> Für die Pankreasverdauung, eine Mischung aus Liberase und Collagenase Typ IV. Verdünnen Liberase TL (Thermo niedrig) 1 Fläschchen 5 mg mit 26 ml DMEM (Dulbeccos modifiziertes Eagle-Medium). </li><li> Vorbereitung Collagenase Typ IV in Hanks-Puffer bei einer Konzentration von 0,5 mg / ml, mit 5 mM HEPES, 0,5 mM CaCl 2, 0,1 mg / ml DNAse und 1 mg / ml Rinderserumalbumin. </li><li> Mischen den Liberase TL und Collagenase-Lösungen in einem Verhältnis von 4: 1 (nach Volumen), das heißt, 4 ml Liberase TL + 1 ml Kollagenase. Aliquot der Mischung von Enzymen zu jedem und bei -20 ° C 2,5 ml. HINWEIS: Die Gewebe Inkubationszeiten vary ein wenig zwischen den Chargen des Enzyms (30 sec bis 1 min-Differenz). Daher kann ein Batch-Test erforderlich. </li></ol></li><li> RPMI Isolation Medien <ol><li> Lösen Sie eine Phiole mit RPMI 1640-Pulver in 1 l Wasser bei Raumtemperatur und zu ergänzen, mit 2 g NaHCO 3, 4,02 g HEPES. </li><li> Kombinieren Sie die RPMI Isolationsmedien in einer großen Erlenmeyerkolben, der mit einem Magnetrührer mischen, auf pH 7,4 mit NaOH, Filter zu sterilisieren und bei 4 ° C. </li></ol></li><li> RPMI Kulturmedien </li><li> Verwenden RPMI Kulturmedium, mit 10% fötalem Rinderserum und 1% Antibiotika (dh, Penicillin 100 U / ml, Streptomycin 100 ug / ml). </li></ol></li><li> Islet Isolation und Kultivierung <ol><li> Tieropfer <ol><li> Opfern Mäuse durch Genickbruch oder CO 2 Ersticken nach Ethik Verfahren der lokalen Institution. Sprühen Sie die Maus Körper gründlich mit 70% Ethanol. </li></ol></li><li> Pancreatic Perfusion <ol><li> Use 2 lange Schnitte seitlich durch die Bauchhaut und Bauchfell, um eine V-förmige Klappe zu machen. Ziehen Sie die Klappe der Haut bis zu den gesamten Bauchraum aufzudecken, beiseite den Darm und die Leber zu halten anstelle von einem gefalteten Seidenpapier, um den Gallengang zu offenbaren (Abbildungen 1 und 2). Setzen Sie das Tier, so dass der Kopf in Richtung der Chirurg und der Schwanz vom Chirurgen. </li><li> Anwenden einer Gefäßklemme an der Ampulle an der Wand, um den Zwölffingerdarm injiziert, Enzyme aus der Eingabe in das Duodenum (Figur 1) zu blockieren. Hinweis: Dies ist ein wichtiger Schritt, da sie bestimmt, ob die injizierten Enzyme werden in den Pankreasgang gehen und durchströmen die Bauchspeicheldrüse oder Rücklauf auf die Injektionsstelle (über Klemme) oder gehen Sie in den Zwölffingerdarm (unter Klemme). </li><li> Kanülieren den Gallengang mit einer 31 G-Nadel in der Nähe der Kreuzung der Ductus hepaticus und cysticus (Abbildung 1) und lassen Raum für eine zweite Injektion, wenn the Pankreas versagt, das erste Mal durchströmen. Langsam (~ 10 sec) zu injizieren etwa 2 ml kaltem Enzymgemisch in die Maus Gallengang. ANMERKUNG: Das Pankreas wird schnell perfundiert, wenn sich die Klammer in der richtigen Position ist. </li><li> Stellen Sie die Klemmposition, wenn die Nadel in den Gallengang, sondern kann die Bauchspeicheldrüse nicht Perfusion (dh., Nach oben die Klammer ein wenig weg von der Leber, wenn überspannt oder verschieben Sie sie ein wenig nach unten in die Leber, wenn im Rahmen eingespannt). HINWEIS: Die Nadel geht manchmal in die umgebende Kapsel eher als das Lumen der Leitung. Eine sinnvolle Zeichen zu überprüfen, ob die Nadel innerhalb der Gallengang ist die Erweiterung des Kanals bei der Injektion. Wenn die Bauchspeicheldrüse nicht zu durchströmen, ist eine Alternative die Enzymmischung konnte auf mehrere Standorte der Bauchspeicheldrüse mit geringeren Ausbeute erwartet injiziert werden. Die Perfusion der linken Seite der Bauchspeicheldrüse (Lobus Splenicus) ist wichtig für die Maximierung der Inselausbeute. </li><li> Nachdem der Bauchspeicheldrüse ist perfused, schnell die Bauchspeicheldrüse zu entfernen, legen Sie sie in einem Fläschchen (~ 20 ml Glas) und Inkubation für ~ 19 min in einem 37 ° C Wasserbad. HINWEIS: Die Inkubationszeit kann ein wenig abhängig von der Mausstamm und Alter variieren. </li><li> Nach der Inkubationszeit, stoppen die Verdauung durch Zugabe von ~ 20 ml kaltem Isolationsmedien. Schütteln Sie die Durchstechflasche vorsichtig, um die Bauchspeicheldrüse zu stören und gießen Sie sie durch eine normale Teesieb (Porendurchmesser von 1 mm) in ein steriles 50 ml Tube. Füllen Sie das Rohr mit Isolierung Medien und Zentrifuge bei 400 xg für 1 min bei 4 ° C mit Bremse ab. </li><li> Lösen Sie das Pellet in der Isolierung Medium erneut und übertragen, um zwei 15-ml-Röhrchen, dann bei 400 xg zentrifugieren für 1 min bei 4 ° C mit Bremse ab. Nach dem Absaugen der Überstand, mischen Sie die beiden Pellets auch mit 6 ml Dichtegradient Zellseparationsmedium (Histopaque 1077 Lösung) pro Röhrchen durch Vortexen oder Auf-und Abpipettieren. Dann vorsichtig hinzu ~ 6 ml der Isolierungsmedien zur Seite des Rohres überDer Dichtegradient Trennmedien und zentrifugiert bei 100 g für 15 min bei 4 ° C mit Bremse ausgeschaltet. </li><li> Sammeln Sie den Überstand in der oberen (Isolationsmedien), mittleren und unteren (Dichtegradienten Zelltrennmedien) Schicht in 3 separate Küche. Interpreten Sie die Inseln in diesen 3 Gerichte, mit einer Pipette unter einem Binokular. HINWEIS: Die mittlere Schicht die meisten der Inseln enthält. Inseln werden als kompakte Strukturen von ~ 50 bis 500 um in der Größe (Figur 3) erkannt wird. Wir erhalten ~ 200 Inseln in WT-Mäusen und 300- &lt;100 Inseln in db / db-Mäusen. </li></ol></li><li> Islet Kultivierung <ol><li> Um die Inseln zu gewinnen aus dem Enzymverdau Kulturgruppen von 40 Inseln in einer 25 cm Petrischale mit 6 ml RPMI-Kulturmedium (10 mM Glukose) bei 37 ° C, 5% / 95% CO 2 / O 2 für 2 Tage vor der Live -Zelle 2-Photonen-Bildgebung. Ändern Sie den Kulturmedien täglich. </li></ol></li></ol></li></ol> 2. Live Cell 2-Photonen-Imaging <ol><li> Vorbereitung der Reagenzien <ol><li> Extrazelluläre Puffer <ol><li> Vorbereitung der extrazellulären Natriumpuffer für die 2-Photonen-Assay mit 140 mM NaCl, 5 mM KCl, 2,5 mM CaCl 2, 1 mM MgCl 2, 5 mM NaHCO 3 und 10 mM HEPES. </li></ol></li><li> Extrazelluläre Dye <ol><li> Für die 2-Photonen-Test vorbereiten stock SRB 8 mM in extrazellulären Puffer, aliquoten in kleine Rohre und bei -20 ° C. Von der Stamm SRB 1:10 in frischem extrazellulären Puffer auf eine Arbeitskonzentration von 0,8 mm zu erhalten. </li></ol></li></ol></li><li> Photonen-Imaging <ol><li> Erste, vorge inkubieren 2 Tage kultivierte Inseln in 3 mM Glucose extrazelluläre Puffer für 30 min. HINWEIS: Wir verwenden Inselchen zwischen 2 und 5 Tagen in Kultur. </li><li> Dann legen Sie eine Objektträger auf die auf dem Mikroskoptisch montiert thermo-Steuerkammer. Verwenden Sie Fett auf das Austreten von Lösung zu verhindern. </li><li> Decken Sie die Kammer mit 500 ul extradere Puffer, enthaltend 0,8 mM SRB mit unterschiedlichen Glukosekonzentrationen (3 mm, 6 mm, 15 mm und 20 mM Glucose). </li><li> Als nächstes legen Sie die vorinkubierten kleine Insel in der Mitte der Kammer und konzentrieren sich auf 3-4 Zellschichten in der kleinen Insel, wo, in Nagetier Inselchen, Immunfärbung für Insulin zeigt, dass die meisten Zellen sind beta-Zellen. HINWEIS: Die SRB alle die Zellmembran der Insel schnell zu skizzieren und die kleine Insel ist bereit, image. </li><li> Verwenden Sie eine 2-Photonen-Mikroskop mit einem 60x Ölimmersionsobjektiv (NA 1,42) und Imaging-Software (z. B. scanimage 11). Abbildung 4 zeigt eine typische Anordnung Mikroskopie. Bild Exozytose-Ereignisse mit SRB als Membran impermeante Fluoreszenzextrazellulären Marker durch Femtosekundenlaserpulsen bei 880 nm angeregt, mit Fluoreszenz bei 550-650 nm detektiert. Notieren Sie die Antworten auf die Insel Stimulation. </li><li> Identifizieren Sie einzelne Exozytose-Ereignisse durch das plötzliche Erscheinen eines kleinen fluoreszierenden Flecks (Auflösung 10 Pixel /1, m) und bestätigen Sie mit der schnell ansteigenden Phase des Fluoreszenzsignals über einen Bereich von Interesse (~ 0,78 um 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.

Der wichtigste Faktor für die Inselisolierung ist die anfängliche Durchblutung des Pankreas; ein unter perfundierten Pankreas führt zu einem erheblich niedrigeren Inselausbeute. Andere Faktoren, die auch Auswirkungen auf die Isolierung Qualität wie die Aufschlusszeit und der Höhe der Erschütterung, die teilweise für die Höhe der Durchblutung zu kompensieren könnte. Zum Beispiel wird ein voll durchströmten Bauchspeicheldrüse müssen bei 37 ° C für ~ 18 min 30 sec inkubiert werden unter leichtem Schütteln im...

Die Rolle der Betazelldysfunktion bei Krankheiten ist allgemein anerkannt, 3,4. Zelllinien, wie der MIN6 und INS-1 sind nützliche Werkzeuge, um die Biologie von Beta-Zell-Verhalten zu verstehen. Allerdings ist die physiologische Kontrolle der Insulinsekretion findet innerhalb der Langerhans-Inseln. Diese Inseln enthalten Tausenden eng gepackten Beta-Zellen, sowie Blutgefäße und anderen endokrinen Zelltypen. Diese Umgebung innerhalb des Insel beeinflusst Insulinsekretion und dürfte bei Diabetes wichtig. Um daher die physiologischen Kontrolle der Insulinsekretion und die Pathophysiologie der Erkrankung zu verstehen, ist es wichtig, intakte Inseln studieren. Inselisolierung Live menschlichen Inselchen und insbesondere menschlichen Inselzellen vom Typ 2-Diabetes-Patienten sind schwer zu bekommen. Darüber hinaus haben die menschliche Inselzellen begrenzte Möglichkeiten für experimentelle molekulare Manipulation. Forscher haben daher eingesetztekönnen aus Tieren und Tiermodellen des Typ 2 Diabetes. Ein solcher Krankheitsmodell ist der db / db-Maus. Dies ist eine spontane Mutation, die Models Typ-2-Diabetes mit einem Phänotyp Progression, die menschliche Krankheit 5,6 eng Parallelen. Die für die Inselisolierung aus diabetischen db / db Mäusen hier vorgestellten Protokoll hat viele Schritte gemeinsam mit anderen Gruppen mit einigen Verfeinerungen für bessere Ausbeute, Reinigung und verbesserte Insellebens. 2 Photonen-Imaging Die Live-cell 2-Photonen-Assay hier beschriebenen ermöglicht es Forschern, um die Anzahl zu quantifizieren und zu bewerten die Merkmale der einzelnen insulinhaltigen Granulat aus vielen Zellen der diabetischen 7 und Wildtyp-Inselchen 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

Typ 2-Diabetes ist eine chronische Krankheit, die 382 Millionen Menschen im Jahr 2013 und wird voraussichtlich bis zu 592 Mio. im Jahr 2035 1 steigen. In den letzten 2 Jahrzehnten die Rolle der Beta-Zell-Dysfunktion bei Typ 2 Diabetes wurde klar festgelegt 2. Forschung Fortschritte Methoden zur Isolierung von Langerhans-Inseln erforderlich. Das Protokoll der Inselisolierung hier präsentierten viele gemeinsame Schritte mit Protokollen von anderen Gruppen, mit einigen Änderungen, um die Ausbeute und Qualität der isolierten Inseln sowohl aus dem Wildtyp und diabetischen Lepr db (db / db) Mäusen zu verbessern. Ein Live-Zelle der 2-Photonen-Abbildungsverfahren wird dann präsentiert, die verwendet werden können, um die Kontrolle der Insulinsekretion in Inseln zu untersuchen.

Islet Ausbeute und Reinigung Für eine normale Wildtyp-Maus, werden etwa 200 kleine Inseln erwartet. Gesunde Inseln aussehen hell, rund geformt und haben einen glatten Rand. Eine Über verdaut Isolierung Charge hat in der Regel klein und Fuzzy-Inseln, während ein Unter verdaut Charge hat weniger kleine Inseln und Inselchen Azinuszelltumoren angebracht (Abbildung 3). Für diabetischen db / db-Mäuse, die kleine Insel Ausbeute und Aussehen abhängig von der Krankheitsprogress...

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; Mausmodell des Typ-2-Diabetes: Pancreatic Islet Isolation und Live-cell 2-Photonen-Imaging von intakten Inseln

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

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 Aufrufe.  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.

Serie: 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.

Isolierung humaner Inselzellen aus teilweise Pankreatektomierten

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

Forschung

Medizin

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.

Färbeprotokollen for Human Pankreasinseln

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.

Quantifizierung glomeruläre Permeabilität von fluoreszierenden Makromoleküle Verwendung von 2-Photonen-Mikroskopie in München Wistar Ratten

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

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.

Isolierung, Kultur und Imaging von humanen fötalen Pankreas-Zellhaufen

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.

Dynamic Contrast Verbesserte Spintomographie von einem orthotopen Mausmodell Bauchspeicheldrüsenkrebs

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.

Chirurgische Verletzung des Maus-Pankreas durch Ligatur des Pankreasganges als Modell für endokrine und exokrine Reprogrammierung und 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 ...

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 Transplantation der Bauchspeicheldrüse Inselchen im Mausmodell

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.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

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.

Längsschnitt in Vivo Bildgebung und Quantifizierung der menschlichen Pankreas-Islet-Grafting- und Beitragenden Wirtszellen in der Vorderen Augenkammer

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

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Mit der 2-Photonen-Mikroskopie die Auswirkungen einer chronischen einseitigen Harnleiterobstruktion auf glomeruläre Prozesse quantifizieren

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...