Ce travail a été soutenu par des subventions du NIH DK078732 (à PTF).

1. Transduction adénovirale et la culture d&#39;îlots de rats <ol><li> Préparer une plaque à 6 puits de culture tissulaire non revêtu par addition de 2 ml de milieu (RPMI 1640 contenant 8 mM de glucose, 10% de sérum bovin foetal, de 50 unités / ml de pénicilline, et 50 mg / ml de streptomycine) au nombre requis de puits. Par exemple, une expérience typique peut exiger trois puits - un de chaque pour un contrôle sans virus, un contrôle du virus (par exemple, GFP-exprimer adénovirus), et le groupe expérimental. </li><li> Chauffer la plaque à 37 ° C en le plaçant dans un incubateur de culture tissulaire pendant au moins 30 min. </li><li> Immédiatement après isolement des îlots de rat 18,19, 100-200 îlots lieu dans des puits individuels de la plaque à 6 puits culture non-tissu enduit. Soixante îlots sont nécessaires pour la sécrétion d&#39;insuline et des essais d&#39;incorporation de thymidine. Les îlots restants peuvent être utilisés pour l&#39;isolation de l&#39;ARN pour les études d&#39;expression génique ou l&#39;isolement de protéines pour immunoblot. </li></ol> [Note: De ce point de l&#39;avant, s&#39;il vous plaît suivre les protocoles institutionnels pour la manipulation, l&#39;utilisation et l&#39;élimination des matières infectieuses.] <ol start="4"><li> Agiter doucement la plaque pour amener les îlots au centre du puits. </li><li> Pipeter l&#39;adénovirus directement sur les îlots dans le centre du plat. Utilisez 100-500 multiplicités d&#39;infection (MOI, le rapport des cellules cibles à virales unités formatrices de plages). </li><li> Laisser reposer pendant 5 min îlots. </li><li> Placer la plaque dans l&#39;incubateur de culture tissulaire (37 ° C, 5% de CO 2). </li><li> Après 24 h, agiter doucement la plaque pour amener les îlots aux centres des puits et transférer les îlots en utilisant une micropipette P200 à un nouveau puits contenant du milieu frais. Si les îlots sont attachés à la plaque, ils peuvent être délogés en douceur avec la pointe de la pipette. </li></ol> [Remarque: Pour vérifier effi transduction adéquatecy, l&#39;utilisation d&#39;un virus de la GFP de commande exprimant est bénéfique, comme îlots peut alors être imagée par microscopie confocale à vérifier la pénétration de l&#39;adénovirus dans le noyau îlot.] <ol start="9"><li> Culture des îlots pour un supplément de 24-72 heures, selon le rythme souhaité de l&#39;expérience des études pilotes d&#39;optimisation. Par exemple, l&#39;induction d&#39;une réponse proliférative peut exiger des durées allant de 24 à 72 h ou knockdown du gène d&#39;intérêt peut exiger 48 ou 72 heures. Transfert des îlots sur des milieux frais chaque jour. </li><li> Pour la finale 24 h de l&#39;expérience, la culture des îlots dans un milieu contenant 1 pCi [méthyl-3 H] -thymidine/ml médias (généralement de 1 pl thymidine / ml les médias). </li></ol> [Note: De ce point de l&#39;avant, s&#39;il vous plaît suivre les protocoles institutionnels pour la manipulation, l&#39;utilisation et l&#39;élimination des matières radioactives.] 2. Essai sécrétion d&#39;insuline <ol><li> Préparer letampon de dosage sécrétion (SAB) solution stock 10X (1,14 M de NaCl, 47 mM de KCl, 12 mM KH 2 PO 4, 11,6 mM MgSO 4) et solution de CaCl 2 de stock 100X (0,25 M CaCl 2). Ces solutions mères peuvent être préparées à l&#39;avance et conservé à température ambiante. </li><li> Fraîchement préparer 50 ml de la RFA de travail (5 mL de SAB 10X, 1 ml de HEPES 1 M, 0,5 ml de CaCl 2, 100X 0,28 ml de 35% de BSA, 0,11 g de NaHCO 3, et de l&#39;eau stérile à 50 ml) dans un conique de 50 ml tube et chaude à 37 ° C en plaçant dans un bain-marie à 37 ° C. </li><li> Introduire à la pipette 10 ml de la SAB de travail dans un tube de 15 ml conique et ajouter 66,8 ul de 2,5 M de D-glucose pour préparer le taux élevé de glucose (16,7 mM) SAB. </li><li> Ajouter 44,8 ul de 2,5 M de D-glucose à la tranche restante de 40 ml de la SAB travaille pour préparer le faible taux de glucose (2,8 mM) SAB. </li><li> Étiquette trois microtubes de 1,7 ml pour chaque puits de la plaque à 6 puits et ajouter 1 ml de tampon phosphate salin (PBS). </li></ol&gt; [Remarque: Comme les îlots sont radioactifs, s&#39;il vous plaît suivre les protocoles institutionnels pour la manipulation, l&#39;utilisation et l&#39;élimination des matières radioactives.] <ol start="6"><li> Placez 20 îlots dans chaque microtube. Faites de chaque tentative d&#39;ajout d&#39;îlots de taille comparable à chaque microtube. Par exemple, chaque tube peut contenir 5 à petite, 10 moyennes et 5 de grande taille (îlots voir Figure 1). </li></ol> [Note: Les îlots peuvent être visualisées en utilisant soit un stéréoscope dissection ou d&#39;un microscope standard.] <ol start="7"><li> Après les îlots se sont installés sur le fond du tube par gravité (~ 2 min), aspirer le PBS avec une micropipette et le jeter. </li></ol> [Remarque: Comme une alternative à la décantation par gravité, les tubes peuvent être centrifugés à 300 g pendant 1 min.] <ol start="8"><li> Pour la pré-incubation, ajouter 400 ul de la faible glucose SAB, placer les tubes (avec leurs bouchons ouvert) dans la incubateur de culture tissulaire (37 ° C, 5% de CO 2), et pré-incuber pendant 60 min. Aspirer le pré-incubation faible taux de glucose SAB et le jeter. </li><li> Pour la sécrétion d&#39;insuline basale, ajouter 400 ul de la faible concentration de glucose SAB, placer les tubes (avec leurs bouchons ouvert) dans la incubateur de culture tissulaire (37 ° C, 5% de CO 2), et incuber pendant 60 min. Recueillir le faible taux de glucose SAB et enregistrer pour le dosage radioimmunologique l&#39;insuline. </li><li> Pour la sécrétion d&#39;insuline stimulée, ajouter 400 ul de la forte concentration de glucose SAB, placer les tubes (avec leurs bouchons ouvert) dans la incubateur de culture tissulaire (37 ° C, 5% de CO 2), et incuber pendant 60 min. Recueillir le taux élevé de glucose SAB et enregistrer pour le dosage radioimmunologique l&#39;insuline. </li></ol> 3. Essai d&#39;incorporation de thymidine <ol><li> Ajouter 1 ml de PBS, après les îlots se sont installés sur le fond du tube par gravité, aspirer le PBS avec une micropipette, jeter, et répétez cettel&#39;étape la fois. </li><li> Ajouter 500 ul de la glace froide d&#39;acide trichloracétique (TCA, 10% w / v) et incuber sur de la glace pendant 30 min. </li><li> Centrifuger les tubes à 16 000 xg pendant 3 min à 4 ° C. </li><li> Aspirer le TCA, ajouter 80 ul de NaOH 0,3 N, et incuber pendant 30 min à température ambiante. Pendant ce temps, vigoureusement vortex les échantillons pour 5-10 s toutes les 10 min. </li><li> Ajouter 4 ml de cocktail de comptage Econo-sécuritaire de 7 ml de liquide à scintillation tubes de comptage. </li><li> Ajouter 50 ml de l&#39;échantillon dans le tube de comptage à scintillation, plafonner le tube, agiter brièvement, et compter dans un compteur à scintillation liquide. </li><li> Mesurer la concentration de protéines en utilisant l&#39;acide bicinchoninique (BCA) de dosage et 10 ul de l&#39;échantillon selon le protocole du fabricant. </li></ol> 4. Analyse des données <ol><li> Effectuer le dosage radioimmunologique l&#39;insuline en suivant le protocole du fabricant. </li><li> Normaliser la sécrétion d&#39;insuline et des données d&#39;incorporation de thymidine avec le concentré de protéineentration. </li></ol> 5. Les résultats représentatifs Un exemple de l&#39;expérience pour évaluer la réplication des îlots et la fonction des cellules bêta des îlots de Langerhans chez le rat est montré dans la figure 2. Cet exemple montre que la surexpression adénovirale de hypothétique &quot;Gene # 6&quot; stimule la réplication robuste îlot sans altérer la fonction des cellules bêta. Dans le panneau supérieur, les résultats du test d&#39;incorporation de thymidine démontrer que l&#39;augmentation de l&#39;expression de &quot;Gene # 6&quot; augmente la synthèse d&#39;ADN, telle que mesurée par l&#39;incorporation de thymidine. Parce que la plupart des cellules de l&#39;îlot de rat sont des cellules bêta, il est probable que cette augmentation de l&#39;incorporation de thymidine indique une augmentation de la réplication des cellules bêta. Cependant, les expériences de confirmation doit être effectuée pour établir fermement cette. Dans le panneau du bas, les résultats du test la sécrétion d&#39;insuline démontrer que la surexpression de &quot;Gene # 6&quot; ne modifie pas l&#39;une des fonctions primaires de cellules bêta, c&#39;est à dire, ila sécrétion de glucose nsulin à basse et haute. La qualité de l&#39;isolement des îlots et la santé des îlots suivant le traitement avec des adénovirus est indiqué par la multiplication par la sécrétion d&#39;insuline à des concentrations de glucose basse et haute. Si l&#39;augmentation de l&#39;expression de &quot;Gene # 6&quot; fonction des cellules bêta avec facultés affaiblies, ce serait probablement correspond à une diminution de l&#39;insuline sécrétée à fortes concentrations de glucose (16,7 mM de stimulation). Une courbe dose-réponse pour différentes concentrations de glucose pourrait également être effectuée. <img src="/files/ftp_upload/4080/4080fig1.jpg" alt="Figure 1"/> Figure 1. Vue d&#39;ensemble du protocole pour évaluer la réplication des îlots et la fonction des cellules bêta des îlots de Langerhans isolés de rat après adéno-médiation des altérations dans l&#39;expression des gènes. Îlots de rat fraîchement isolés sont exposés à des adénovirus pendant 24 h puis cultivées jusqu&#39;à 96 h. L&#39;incorporation de thymidine est évalué dans le dernier 24 h, suivi par la mesure de la sécrétion d&#39;insuline àglucose basse et haute. <img src="/files/ftp_upload/4080/4080fig2.jpg" alt="Figure 2"/> Figure 2. Les résultats d&#39;une expérience en utilisant un adénovirus contrôle et un adénovirus un gène hypothétique étiquetés comme &quot;Gene # 6&quot; surexprimant. Le panneau supérieur montre l&#39;incorporation de la thymidine et le panneau inférieur de la sécrétion d&#39;insuline.

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Pas de conflits d&#39;intérêt déclarés.

Établir des voies qui peuvent être modulés afin de stimuler la réplication et d&#39;améliorer la fonction des cellules bêta sont pertinentes pour les deux principales formes de diabète. Parce que fonctionnelle masse des cellules bêta est tributaire de l&#39;existence et la fonction de cellules sécrétrices d&#39;insuline, l&#39;évaluation de ces déterminants a simultanément ses avantages. Ce protocole décrit un protocole simplifié pour déterminer si la surexpression ou la suppression d&#39;une protéine c...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> RPMI 1640 les médias </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> Pénicilline / streptomycine </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> Des plaques 6 puits </td><td> BD-Falcon </td><td> 35-1146 </td><td> Non-TC traités </td></tr><tr><td> [Méthyl-3 H]-thymidine </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Micro-centrifugeuse tubes </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> Solution à 1 M </td></tr><tr><td> 35% de 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% p / v </td></tr><tr><td> NaOH </td><td> Acros </td><td> 12426 </td><td></td></tr><tr><td> Tube de comptage à scintillation </td><td> Sarstedt </td><td> 58.536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Bouchon du tube de comptage à scintillation </td><td> Sarstedt </td><td> 65.816 </td><td></td></tr><tr><td> Econo-Safe cocktail de comptage </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> L&#39;insuline 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> Équipement </td></tr><tr><td> Centrifuger </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Comptage à scintillation portoir </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Compteur à scintillation liquide </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

L&#39;homéostasie du glucose est principalement contrôlée par les hormones du système endocrinien de l&#39;insuline et le glucagon, sécrété par la version bêta du pancréas et des cellules alpha, respectivement. Fonctionnelle masse des cellules bêta est déterminée par la masse des cellules bêta anatomique ainsi que la capacité des cellules bêta pour répondre à une charge en éléments nutritifs. Une perte de masse des cellules bêta fonctionnelles est au cœur de ces deux formes principales de diabète 1-3. Considérant que les résultats en déclin bêta fonctionnelles de masse de cellules à partir d&#39;une attaque auto-immune de type 1 du diabète, dans le diabète de type 2, cette décroissance se développe à la fois une incapacité des cellules bêta à sécréter de l&#39;insuline de façon appropriée et la destruction des cellules bêta à partir d&#39;un cadre de mécanismes. Ainsi, les efforts visant à restaurer la fonctionnalité de masse des cellules bêta sont primordiaux pour un meilleur traitement des cures et potentiels pour le diabète. Des efforts sont en cours pour identifier les voies moléculaires qui peuvent être exploitées afin de stimuler la réplication et d&#39;améliorer la fonction des cellules bêta.Idéalement, les objectifs thérapeutiques permettrait d&#39;améliorer à la fois la croissance des cellules bêta et la fonction. Peut-être plus important, cependant, c&#39;est de déterminer si une stratégie qui stimule la croissance des cellules bêta se fait au prix de la fonction des cellules bêta atteinte (par exemple avec certains oncogènes) et vice-versa. Par systématiquement la suppression ou la surexpression de l&#39;expression de gènes cibles dans les îlots isolés de rat, on peut identifier des cibles thérapeutiques potentielles pour augmenter la masse des cellules bêta fonctionnelles 4-6. Des vecteurs adénoviraux peut être utilisé à des protéines efficacement surexpriment ou démontable en îlots isolés de rat 4,7-15. Ici, nous présentons une méthode pour manipuler l&#39;expression des gènes en utilisant transduction adénovirale et d&#39;évaluer la réplication des îlots et la fonction des cellules bêta des îlots de Langerhans isolés de rat (figure 1). Cette méthode a été utilisée précédemment pour identifier de nouvelles cibles qui modulent la réplication des cellules bêta ou la fonction 5,6,8,9,16,17.

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

Ce protocole permet d&#39;identifier les facteurs qui modulent fonctionnelle masse des cellules bêta de trouver des cibles thérapeutiques potentielles pour le traitement du diabète. Le protocole consiste en une méthode simplifiée pour évaluer la réplication des îlots et la fonction des cellules bêta des îlots de Langerhans isolés de rat après la manipulation de l&#39;expression des gènes par des adénovirus.

Les îlots de rongeurs évaluation de la réplication et la fonction des cellules bêta dans Adenovirally transduites isolées

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

assessing-replication-beta-cell-function-adenovirally-transduced

Research

JoVE Journal

Medicine

13.9K vues.  Indiana University School of Medicine. Ce protocole permet d'identifier les facteurs qui modulent fonctionnelle masse des cellules bêta de trouver des cibles thérapeutiques potentielles pour le traitement du diabète. Le protocole consiste en une méthode simplifiée pour évaluer la réplication des îlots et la fonction des cellules bêta des îlots de Langerhans isolés de rat après la manipulation de l'expression des gènes par des adénovirus.

Vidéo: 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.

L&#39;évaluation de la fonction cardiaque et Energétique dans les coeurs isolés de souris à l&#39;aide<sup&gt; 31</sup&gt; P NMR Spectroscopy

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

Recherche

Médecine

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.

Évaluer la fonction endothéliale Vasodilatateur avec le 2000 Endo-PAT

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-mécanique Analyse des muscles squelettiques isolés

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.

L&#39;isolement des îlots humains à partir des patients partiellement pancréatectomisés

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.

Évaluer les changements tératogènes dans un modèle de poisson zèbre de l&#39;exposition d&#39;alcoolisation fœtale

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.

Protocoles de coloration pour des îlots pancréatiques humains

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; Modèle de souris de diabète de type 2: pancréatique isolement des îlots et des cellules vivantes 2-Photon Imaging d&#39;îlots intacts

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.

Un haut contenu<em&gt; In vitro</em&gt; Pancreatic Islet β-cellulaire Plateforme de découverte de la réplication

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.

3D quantitative<em&gt; In Silico</em&gt; Modélisation (q3DISM) de Cerebral bêta-amyloïde phagocytose dans les modèles rongeurs de la maladie d&#39;Alzheimer

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.

Préservation des nerfs Obstruction à mi-urétrale (NEMO) chez les femelles petits rongeurs

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