Diese Arbeit wurde von DK078732 Zuschuss von der NIH (bis PTF) unterstützt.

1. Adenovirale Transduktion und Kultivierung von Ratteninseln <ol><li> Vorbereiten einer 6-Well-Gewebekultur nicht beschichteten Platte durch Zugabe von 2 ml Medium (RPMI 1640 Medium mit 8 mM Glukose, 10% fötales Rinderserum, 50 Einheiten / ml Penicillin und 50 ug / ml Streptomycin), um die erforderliche Anzahl von Brunnen. Zum Beispiel kann ein typisches Experiment erfordert drei Vertiefungen - jeweils für einen nicht-Viruskontrolle, ein Virus (zB, GFP-exprimierende Adenovirus), und die experimentelle Gruppe. </li><li> Erwärmen Sie die Platte auf 37 ° C, indem Sie ihn in eine Gewebekultur-Inkubator für mindestens 30 min. </li><li> Unmittelbar nach Ratte Inselisolierung 18,19, Platz 100-200 Inselchen in einzelnen Wells der 6-well Non-Gewebekultur beschichteten Platte. Sechzig Inseln sind für die Insulinsekretion und Thymidineinbau Assays erforderlich. Die übrigen Inseln können zur RNA-Isolierung für Untersuchungen der Genexpression oder Proteinisolierung zum Immunoblotting verwendet werden. </li></ol> [Hinweis: Von diesem Punkt an, folgen Sie bitte institutionellen Protokolle für die Behandlung, Verwendung und Entsorgung biologischer Gefahrenstoffe.] <ol start="4"><li> Die Platte vorsichtig schwenken, um die kleinen Inseln in die Mitte des Brunnens zu bringen. </li><li> Pipettieren Sie die Adenovirus direkt auf den kleinen Inseln in der Mitte des Tellers. Verwenden 100-500 Multiplizitäten der Infektion (MOI, das Verhältnis von Zielzellen, um virale Plaque-bildenden Einheiten). </li><li> Lassen Sie die kleinen Inseln Rest für 5 min. </li><li> Die Platte wird in der Gewebekultur-Inkubator (37 ° C, 5% CO 2). </li><li> Nach 24 h wurde die Platte vorsichtig schwenken, um die Inseln zu den Zentren der Brunnen bringen und übertragen Sie die kleinen Inseln mit einer Mikropipette P200 zu einem neuen Brunnen mit frischem Medien. Wenn die Inseln an der Platte werden, können sie leicht mit der Pipettenspitze abgelöst werden. </li></ol> [Hinweis: Um eine ausreichende Transduktion Effizienz überprüfency, die Verwendung eines Kontrollvirus GFP exprimieren vorteilhaft ist, als Inseln kann dann über der konfokalen Mikroskopie, um das Eindringen des Adenovirus in der Insel Kern zu überprüfen abgebildet werden.] <ol start="9"><li> Kultur die Inseln für eine zusätzliche 24-72 h, je nach dem gewünschten Timing des Experiments von der Optimierung Pilotstudien. Zum Beispiel kann Induktion einer proliferativen Antwort erfordern Zeiten im Bereich von 24-72 h oder Herunterregulierung von dem Gen von Interesse kann 48 oder 72 Stunden benötigen. Übertragen Sie die Inseln an die frische Medien jeden Tag. </li><li> Für die letzten 24 h des Experiments, Kultur der Inseln in einem Medium, 1 Ci [Methyl-3 H] -thymidine/ml Medien (in der Regel 1 ul Thymidin / ml Medium). </li></ol> [Hinweis: Von diesem Punkt an, folgen Sie bitte institutionellen Protokolle für die Behandlung, Verwendung und Entsorgung von radioaktivem Material.] 2. Insulin-Sekretion-Assay <ol><li> Bereiten Sie dieSekretion-Assay-Puffer (SAB) 10X-Stammlösung (1,14 M NaCl, 47 mM KCl, 12 mM KH 2 PO 4, 11,6 mM MgSO 4) und CaCl 2 100x Stocklösung (0,25 M CaCl 2). Diese Stammlösungen können im Voraus geplant werden und bei Raumtemperatur gelagert. </li><li> Frisch zubereiten 50 ml des Arbeits-SAB (5 ml 10X SAB, 1 ml 1 M HEPES, 0,5 ml 100X CaCl 2, 0,28 ml 35% BSA, 0,11 g NaHCO 3 und sterilem Wasser auf 50 ml) in einem 50-ml konischen Röhrchen und warm bis 37 ° C, indem man in einem 37 ° C im Wasserbad. </li><li> 10 ml der Arbeits-SAB in einem 15-ml-Erlenmeyerkolben eingewogen und mit 66,8 ul 2,5 M D-Glucose, um die hohe Glucose (16,7 mM) SAB vorzubereiten. </li><li> In 44,8 ul 2,5 M D-Glucose, um die restlichen 40 ml der Arbeitslösung SAB, um die niedrige Glukose (2,8 mm) SAB vorzubereiten. </li><li> Beschriften Sie drei 1,7-ml-Röhrchen für jede Vertiefung der Platte mit 6 Vertiefungen und 1 ml Phosphat-gepufferter Salzlösung (PBS). </li></ol&gt; [Hinweis: Da die Inseln radioaktiv sind, folgen Sie bitte institutionellen Protokolle für die Behandlung, Verwendung und Entsorgung von radioaktivem Material.] <ol start="6"><li> Platz 20 Inselchen in jedes Mikrozentrifugenröhrchen. Machen jeden Versuch, vergleichbar große Inselchen, um jeden Mikrozentrifugenröhrchen hinzuzufügen. Zum Beispiel kann jedes Rohr enthalten 5 kleine, 10 mittlere und 5 großformatige Inseln (siehe Abbildung 1). </li></ol> [Anmerkung: Islets visualisiert werden kann entweder mit einem Stereoskop sezieren oder einen Standard-Mikroskop.] <ol start="7"><li> Nachdem die Inseln auf der Unterseite des Rohres durch die Schwerkraft (~ 2 min) gelöst ist, absaugen PBS mit einer Mikropipette und zu verwerfen. </li></ol> [Anmerkung: Als Alternative zur Abwicklung durch die Schwerkraft, die Rohre können bei 300 xg für 1 min zentrifugiert werden.] <ol start="8"><li> Für Vorinkubation, fügen Sie 400 ul des niedrigen glucose SAB, stellen Sie die Rohre (mit ihren Mützen offen) in die Gewebekultur-Inkubator (37 ° C, 5% CO 2) und Pre-Inkubation für 60 min. Saugen Sie die Vorinkubation niedrige Glukose SAB und entsorgen. </li><li> Für basale Insulinsekretion, fügen Sie 400 ul des niedrigen Glukose SAB, stellen Sie die Rohre (mit ihren Mützen offen) in die Gewebekultur-Inkubator (37 ° C, 5% CO 2) und Inkubation für 60 min. Sammeln Sie die niedrige Glukose SAB und sparen für die Insulin-Radioimmunoassay. </li><li> Für stimulierte Insulinsekretion, fügen Sie 400 ul der hohen Glukose-SAB, stellen Sie die Rohre (mit ihren Mützen offen) in die Gewebekultur-Inkubator (37 ° C, 5% CO 2) und Inkubation für 60 min. Sammeln Sie die hohe Glukose SAB und sparen für die Insulin-Radioimmunoassay. </li></ol> 3. Thymidineinbau Assay <ol><li> 1 ml PBS; nachdem die Inseln auf dem Boden des Rohres durch die Schwerkraft niedergelassen haben, saugen Sie den PBS mit einer Mikropipette, zu verwerfen, und wiederholen Sie dieseSchritt einmal. </li><li> Fügen Sie 500 ul eiskalter Trichloressigsäure (TCA, 10% w / v) und Inkubation auf Eis für 30 min. </li><li> Zentrifugieren bei 16 000 × g für 3 min bei 4 ° C </li><li> Saugen Sie das TCA, fügen Sie 80 ul 0,3 N NaOH, und inkubieren Sie für 30 Minuten bei Raumtemperatur. Während dieser Zeit kräftig Wirbel die Proben für 5-10 Sekunden alle 10 min. </li><li> 4 ml Econo-sichere Zählen Cocktail bis 7 ml Flüssigkeitsszintillationszählung Röhren. </li><li> Dann werden 50 ul der Probe auf die Szintillationszählung Rohr, Röhrchen verschließen, kurz schütteln, und zählen in einem Flüssigkeits-Szintillationszähler. </li><li> Messung der Proteinkonzentration unter Verwendung des Bicinchoninsäure (BCA)-Probe und 10 ul Probe gemäß dem Protokoll des Herstellers. </li></ol> 4. Datenanalyse <ol><li> Führen Sie die Insulin-Radioimmunoassay nach dem Protokoll des Herstellers. </li><li> Normalisieren der Insulinsekretion und Thymidineinbau Daten mit dem Protein Konzentration. </li></ol> 5. Repräsentative Ergebnisse Ein Beispiel für das Experiment zu Insel Replikation und Beta-Zell-Funktion in Ratteninseln Beurteilung wird in 2 gezeigt. Dieses Beispiel zeigt, dass die adenovirale Überexpression von hypothetischen &quot;Gene # 6&quot; robust stimuliert Inselchen Replikation ohne dass Beta-Zellfunktion. In der oberen Platte, zeigen die Ergebnisse aus der Thymidin-Einbau-Assay dass eine Erhöhung der Expression von &quot;Gene # 6&quot; erhöht DNA-Synthese, wie durch den Einbau von Thymidin gemessen. Da die meisten der Zellen in der Ratte Beta-Inselzellen sind, ist es wahrscheinlich, dass dieser Anstieg der Thymidin-Einbau eine Erhöhung in der Beta-Zell-Replikation zeigt. Allerdings müssen bestätigende Experimente durchgeführt, um fest zu etablieren dies werden. In der Bodenplatte, zeigen die Ergebnisse aus der Insulinsekretion Assay dass die Überexpression von &quot;Gene Nr. 6&quot; ergab keine Änderung eines der primären Beta-Zell-Funktionen, dh insulin Sekretion bei niedrigen und hohen Glucose. Die Qualität der Inselisolierung und die Gesundheit der Inseln nach der Behandlung mit Adenoviren erhält fachen Anstieg der Insulinsekretion bei niedrigen und hohen Glucosekonzentrationen angegeben. Bei Erhöhung der Expression von &quot;Gene # 6&quot; gestörte beta-Zell-Funktion, würde dies wahrscheinlich als eine Abnahme in der Insulin bei hohen, stimulatorische Glucosekonzentrationen (16,7 mM) sezerniert reflektiert werden. Eine Dosis-Wirkungs-Kurve für verschiedene Glucosekonzentrationen könnte auch durchgeführt werden. <img src="/files/ftp_upload/4080/4080fig1.jpg" alt="1"/> 1. Überblick Protokoll Insel Replikation und Beta-Zell-Funktion in isolierten Ratteninseln nach adenoviralen-vermittelten Änderungen in der Genexpression. Frisch isolierte Ratteninseln sind Adenoviren für 24 h ausgesetzt und dann bis zu 96 h kultiviert. Thymidin-Einbau in der letzten 24 h beurteilt, gefolgt von der Messung der Insulinsekretion beiniedrige und hohe Glukose. <img src="/files/ftp_upload/4080/4080fig2.jpg" alt="2"/> 2. Ergebnisse aus einem Experiment unter Verwendung eines Kontroll-Adenovirus und ein Adenovirus-Überexpression eines hypothetischen Gens als &quot;Gene # 6&quot; bezeichnet. Das obere Feld zeigt das Thymidin-Einbau und die Bodenplatte der Insulinsekretion.

<ol>
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Einrichtung Wege, die moduliert werden, um die Replikation stimulieren und die Funktion der Beta-Zellen können relevant sind beiden Hauptformen von Diabetes. Da funktionale Betazellmasse ist abhängig von der Existenz und Funktion von Insulin-sezernierenden Zellen, die Beurteilung dieser Determinanten gleichzeitig hat seine Vorteile. Dieses Protokoll beschreibt eine optimierte Protokoll zum Identifizieren, ob die Überexpression oder Unterdrückung eines Proteins an Veränderungen in der funktionellen Betazellmasse <em...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> RPMI 1640 Medien </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> Penicillin / Streptomycin </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 6-Well-Platte </td><td> BD-Falcon </td><td> 35-1146 </td><td> Non-TC behandelt </td></tr><tr><td> [Methyl-3 H]-Thymidin </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Mikro-Zentrifugenröhrchen </td><td> Denville </td><td> C2170 </td><td> 1,7 ml </td></tr><tr><td> NaCl </td><td> Sigma </td><td> 59888 </td><td></td></tr><tr><td> KCl </td><td> Acros </td><td> 42409 </td><td></td></tr><tr><td> KH 2 &lt;/ Sub&gt; PO 4 </td><td> Acros </td><td> 20592 </td><td></td></tr><tr><td> MgSO 4 </td><td> Acros </td><td> 41348 </td><td></td></tr><tr><td> CaCl 2 </td><td> Acros </td><td> 34961 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H0887 </td><td> 1 M Lösung </td></tr><tr><td> 35% BSA </td><td> Sigma </td><td> A7979 </td><td></td></tr><tr><td> NaHCO 3 </td><td> Acros </td><td> 42427 </td><td></td></tr><tr><td> d-Glucose </td><td> Sigma </td><td> G8769 </td><td></td></tr><tr><td> TCA </td><td> Fisher Scientific </td><td> SA9410-1 </td><td> 10% w / v </td></tr><tr><td> NaOH </td><td> Acros </td><td> 12426 </td><td></td></tr><tr><td> Szintillationszählung Rohr </td><td> Sarstedt </td><td> 58,536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Szintillationszählung Tubenkappe </td><td> Sarstedt </td><td> 65,816 </td><td></td></tr><tr><td> Econo-Safe Zählen Cocktail </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> Insulin-RIA </td><td> Siemens </td><td> TKIN2 </td><td></td></tr><tr><td> BCA Assay Kit </td><td> Thermo Scientific </td><td> 23250 </td><td></td></tr><tr><td></td><td></td><td></td><td> Ausrüstung </td></tr><tr><td> Zentrifugieren </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Szintillationszählung Röhrchenständer </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Flüssigkeits-Szintillationszähler </td><td> Perkin Elmer </td><td> Tri-Carb 2910TR </td><td></td></tr></tbody></table>

assessing replication and beta cell function in adenovirally-transduced isolated rodent islets

Glukosehomöostase wird hauptsächlich durch die endokrine Hormone Insulin und Glucagon gesteuert, sekretiert von der Pankreas-Beta und Alpha-Zellen. Funktionelle Betazellmasse wird durch die anatomische Betazellmasse sowie der Fähigkeit der Betazellen zu einer Nährstoffbelastung reagieren bestimmt. Ein Verlust der funktionellen Betazellmasse ist zentral für beide Hauptformen des Diabetes 1-3. Während die sinkende funktionale Betazellmasse ergibt sich aus einer Autoimmun-Attacke beim Typ 1 Diabetes, bei Typ 2 Diabetes entwickelt sich dieser Dekrement sowohl aus der Unfähigkeit der Beta-Zellen gegenüber Insulin angemessen absondern und die Zerstörung der Beta-Zellen aus einem Kader von Mechanismen. Daher sind Bemühungen um eine funktionale Betazellmasse wieder vorrangig für die bessere Therapie und mögliche Heilmittel für Diabetes. Es sind Bemühungen im Gange, um molekulare Signalwege, die ausgenutzt, um die Replikation zu stimulieren und verbessern die Funktion der Beta-Zellen identifizieren kann.Im Idealfall würde therapeutische Ziele verbessern sowohl Beta-Zell-Wachstum und Funktion. Vielleicht noch wichtiger ist jedoch zu erkennen, ob eine Strategie, die Beta-Zellen stimuliert das Wachstum auf Kosten der Beeinträchtigung der Beta-Zellen-Funktion (wie mit einigen Onkogene) und umgekehrt wird. Durch die systematische Unterdrückung oder Überexpression der Expression von Zielgenen in isolierten Ratten-Inseln, kann man potenzielle therapeutische Ziele für die Erhöhung funktionale Betazellmasse 4-6. Adenovirus-Vektoren können, um effizient überexprimieren oder Knockdown Proteine ​​in isolierten Ratteninseln 4,7-15 eingesetzt werden. Hier präsentieren wir eine Methode, um die Genexpression Nutzung adenoviralen Transduktion zu manipulieren und zu bewerten Inselchen Replikation und Beta-Zellfunktion in isolierten Ratten-Inseln (Abbildung 1). Diese Methode wurde bereits auf neue Ziele, die Beta-Zell-Vermehrung oder Funktion modulieren 5,6,8,9,16,17 zu identifizieren.

Watch this Scientific Journal Video about Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets at JoVE.com

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Dieses Protokoll erlaubt es, Faktoren, die funktionelle Betazellmasse modulieren, um potentielle therapeutische Ziele für die Behandlung von Diabetes zu finden identifizieren. Das Protokoll besteht aus einem optimierten Verfahren zur Insel Replikation und Beta-Zellfunktion in isolierten Ratteninseln nach Manipulation der Genexpression mit Adenoviren zu beurteilen.

Die Beurteilung der Replikation und Beta-Zellfunktion in adenoviral-transduzierten Isoliert Nager Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

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JoVE Journal

Medicine

13.9K Aufrufe.  Indiana University School of Medicine. Dieses Protokoll erlaubt es, Faktoren, die funktionelle Betazellmasse modulieren, um potentielle therapeutische Ziele für die Behandlung von Diabetes zu finden identifizieren. Das Protokoll besteht aus einem optimierten Verfahren zur Insel Replikation und Beta-Zellfunktion in isolierten Ratteninseln nach Manipulation der Genexpression mit Adenoviren zu beurteilen.

Serie: Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Beurteilung der Herzfunktion und Energetik in isolierten Maus-Herzen mit 31 P-NMR-Spektroskopie

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

Forschung

Medizin

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

Die Beurteilung Endothelial Vasodilator-Funktion mit der Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

Myo-mechanischen Analyse von isolierten Skelettmuskel

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

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 ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Die Beurteilung Teratogene Änderungen in einem Zebrafisch-Modell der Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

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 ...

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

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, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

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, ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Ein hoher Gehalt<em&gt; In Vitro</em&gt; Pancreatic Islet β-Zell-Replikation-Discovery-Plattform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Quantitative 3D<em&gt; In Silico</em&gt; Modellierung (q3DISM) zerebraler Amyloid-beta Phagozytose in Nagetiermodellen der Alzheimer-Krankheit

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Nerve sparende Mittelharnröhrenobstruktion (NEMO) in Female Kleine Nagetiere

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...