Questo lavoro è stato sostenuto dalla concessione DK078732 dal NIH (per PTF).

1. Trasduzione adenovirale e la coltura di isole di ratto <ol><li> Preparare una ben 6-non-tessuto piastra rivestita coltura aggiungendo 2 ml di mezzo (RPMI 1640 contenente 8 mM di glucosio, 10% di siero fetale bovino, 50 unità / ml di penicillina, e 50 ug / ml di streptomicina) al numero richiesto di pozzi. Ad esempio, un tipico esperimento può richiedere tre pozzetti, uno per ogni mancato controllo virus, un controllo virus (ad esempio, GFP-esprimono adenovirus), e il gruppo sperimentale. </li><li> Riscaldare la piastra a 37 ° C ponendo in un incubatore di coltura tissutale per almeno 30 min. </li><li> Immediatamente dopo isolotto isolamento ratto 18,19, posto 100-200 isolotti in pozzetti individuali della ben 6-non-tessuto rivestito piastra di coltura. Sessanta isolotti sono necessarie per la secrezione di insulina e saggi di incorporazione di timidina. Gli isolotti rimanenti possono essere usati per l&#39;isolamento di RNA per studi di espressione genica o isolamento di proteine ​​per immunoblotting. </li></ol> [Nota: Da questo punto in avanti, si prega di seguire i protocolli istituzionali per la manipolazione, l&#39;uso e lo smaltimento dei materiali a rischio biologico.] <ol start="4"><li> Delicatamente il piastra per portare le isolette al centro del pozzo. </li><li> Pipettare l&#39;adenovirus direttamente sulle isole nel centro del piatto. Utilizzare 100-500 molteplicità di infezione (MOI, il rapporto di cellule bersaglio per virali unità formanti placca). </li><li> Lasciare riposare per 5 minuti isolotti. </li><li> Porre la piastra in tessuto incubatore (37 ° C, 5% CO 2). </li><li> Dopo 24 h, roteare delicatamente la piastra per portare le isolette ai centri dei pozzetti e trasferire gli isolotti utilizzando una micropipetta P200 in un pozzetto contenente mezzi freschi. Se le isolette si attaccano alla piastra, possono essere sloggiato delicatamente con la punta della pipetta. </li></ol> [Nota: per verificare efficienze adeguate trasduzioneCY, l&#39;uso di un virus di controllo che esprime GFP è vantaggiosa, in quanto isolotti può quindi essere rappresentata mediante microscopia confocale per verificare la penetrazione del adenovirus nel nucleo isolotto.] <ol start="9"><li> Cultura le isolette per un ulteriore 24-72 ore, a seconda della temporizzazione desiderata dell&#39;esperimento da studi pilota di ottimizzazione. Ad esempio, l&#39;induzione di una risposta proliferativa può richiedere tempi compresi fra 24-72 ore o knockdown del gene di interesse può richiedere 48 o 72 ore. Trasferire le isolette di mezzi freschi ogni giorno. </li><li> Per la finale 24 ore dell&#39;esperimento, la cultura delle isole in un mezzo contenente 1 microCi [metil-3 H] -thymidine/ml media (in genere 1 pl timidina / ml media). </li></ol> [Nota: Da questo punto in avanti, si prega di seguire i protocolli istituzionali per la manipolazione, l&#39;uso e lo smaltimento dei materiali radioattivi.] 2. Secrezione insulinica test <ol><li> Preparare ilsecrezione tampone di saggio (SAB) soluzione madre 10X (1,14 M NaCl, 47 mM KCl, 12 mM KH 2 PO 4, 11,6 mM MgSO 4) e la soluzione di CaCl 2 magazzino 100X (0,25 M CaCl 2). Queste soluzioni fotografici possono essere preparate in anticipo e conservato a temperatura ambiente. </li><li> Appena preparare 50 ml della BRS di lavoro (5 mL di 10X SAB, 1 ml di 1 M HEPES, 0,5 ml di 100X CaCl 2, 0,28 ml di 35% BSA, 0,11 g NaHCO 3, e acqua sterile per 50 ml) in un 50 ml tubo e calda a 37 ° C introducendo in un 37 ° C a bagno-maria. </li><li> Pipettare 10 ml del SAB lavorazione in un 15-ml provetta conica e aggiungere 66,8 pl di 2,5 M D-glucosio per preparare il glucosio (16,7 mM) SAB. </li><li> Aggiungere 44,8 pl di 2,5 M D-glucosio per il restante 40 ml di lavoro SAB per preparare il glucosio bassa (2,8 mM) SAB. </li><li> Label tre 1,7 ml microprovette per ciascun pozzetto della piastra da 6 pozzetti e aggiungere 1 ml di tampone fosfato salino (PBS). </li></ol&gt; [Nota: Poiché le isole sono radioattivi, si prega di seguire i protocolli istituzionali per la manipolazione, l&#39;uso e lo smaltimento dei materiali radioattivi.] <ol start="6"><li> Mettere 20 isolotti in ogni provetta. Fate ogni tentativo di aggiungere isolette di dimensioni relativamente a ciascuna provetta. Ad esempio, ciascun tubo 5 può contenere piccole, medie 10, e 5 grandi dimensioni isolette (vedi Figura 1). </li></ol> [Nota: Le isole possono essere visualizzate utilizzando uno stereoscopio dissezione o un microscopio standard.] <ol start="7"><li> Dopo che le isolette hanno scelto di fondo della provetta per gravità (~ 2 min), aspirare il PBS con una micropipetta e scartare. </li></ol> [Nota: In alternativa alla sedimentazione per gravità, i tubi possono essere centrifugate a 300 xg per 1 min.] <ol start="8"><li> Per la pre-incubazione, aggiungere 400 microlitri della bassa gluglucosio SAB, posizionare i tubi (con i tappi aperto) nel tessuto incubatore (37 ° C, 5% CO 2), e pre-incubare per 60 min. Aspirare il pre-incubazione di glucosio basso SAB ed eliminarla. </li><li> Per la secrezione di insulina basale, aggiungere 400 microlitri del glucosio SAB basso, posizionare i tubi (con i tappi aperto) nel tessuto incubatore (37 ° C, 5% CO 2), e incubare per 60 min. Raccogliere il basso di glucosio SAB e risparmiare per il dosaggio radioimmunologico dell&#39;insulina. </li><li> Per la secrezione di insulina stimolata, aggiungere 400 microlitri del glucosio SAB alto, posizionare i tubi (con i tappi aperto) nel tessuto incubatore (37 ° C, 5% CO 2), e incubare per 60 min. Raccogliere l&#39;alto glucosio SAB e risparmiare per il dosaggio radioimmunologico dell&#39;insulina. </li></ol> 3. Timidina Assay <ol><li> Aggiungere 1 ml di PBS, dopo le isolette hanno scelto di fondo della provetta per gravità, aspirare il PBS con una micropipetta, scartare, e ripetere questapasso una volta. </li><li> Aggiungere 500 pl acido ghiacciata tricloroacetico (TCA, 10% w / v) e incubare su ghiaccio per 30 min. </li><li> Centrifugare le provette a 16 000 xg per 3 min a 4 ° C. </li><li> Aspirare il TCA, aggiungere 80 ul di NaOH 0,3 N, e incubare per 30 min a temperatura ambiente. Durante questo tempo, vigorosamente vortici i campioni per 5-10 s ogni 10 min. </li><li> Aggiungere 4 ml di Econo-safe cocktail contare fino a 7 tubi ml di liquido di scintillazione di conteggio. </li><li> Aggiungere 50 pl di campione al tubo scintillazione, chiudere il tubo, agitare, e conteggio in un contatore a scintillazione liquida. </li><li> Misurare la concentrazione di proteina usando l&#39;acido bicinconinico (BCA) saggio e 10 pl di campione secondo il protocollo del produttore. </li></ol> 4. Analisi dei dati <ol><li> Eseguire il dosaggio radioimmunologico dell&#39;insulina seguendo il protocollo del produttore. </li><li> Normalizza la secrezione di insulina e di dati timidina con il Concentration. </li></ol> 5. Risultati rappresentativi Un esempio di un esperimento per valutare la replicazione delle isole e la funzione delle cellule beta in isole di ratto è illustrato nella figura 2. Questo esempio mostra che l&#39;iperespressione adenovirale di ipotetici &quot;Gene # 6&quot; stimola robusta replica isolotto senza alterare la funzione delle cellule beta. Nel pannello superiore, i risultati del saggio di incorporazione di timidina dimostrare che aumentando l&#39;espressione di &quot;Gene # 6&quot; aumenta la sintesi del DNA, come misurato mediante incorporazione di timidina. Poiché la maggior parte delle cellule del isolotto ratto sono cellule beta, è probabile che questo aumento timidina indica un incremento della replicazione delle cellule beta. Tuttavia, gli esperimenti di conferma devono essere eseguite per stabilire con fermezza questo. Nel pannello inferiore, i risultati del saggio secrezione insulinica dimostrano che la sovraespressione di &quot;Gene # 6&quot; non altera una delle funzioni primarie di cellule beta, cioè, insulin secrezione di glucosio a bassa e alta. La qualità dell&#39;isolamento isolotto e la salute degli isolotti in seguito al trattamento con adenovirus è indicato dalla volte maggiore della secrezione di insulina a basse concentrazioni di glucosio e alta. Se aumentando l&#39;espressione di &quot;Gene # 6&quot; compromissione della funzione delle cellule beta, questo sarebbe probabilmente riflette come una diminuzione dell&#39;insulina secreta a concentrazioni elevate stimolatorie, glucosio (16,7 mM). Una curva dose-risposta per diverse concentrazioni di glucosio potrebbe anche essere eseguita. <img src="/files/ftp_upload/4080/4080fig1.jpg" alt="Figura 1"/> Figura 1. Panoramica del protocollo per valutare la replica delle isole e la funzione delle cellule beta nelle isole di ratto isolate in seguito adenovirale-mediate alterazioni nell&#39;espressione genica. Isole di ratto isolate di fresco sono esposti a adenovirus per 24 ore e poi coltivate fino a 96 h. Timidina viene valutata in finale 24 ore, seguita dalla misura della secrezione di insulinaglicemia bassa e alta. <img src="/files/ftp_upload/4080/4080fig2.jpg" alt="Figura 2"/> Figura 2. Risultati di un esperimento utilizzando un adenovirus di controllo e un adenovirus sovraesprimendo un gene ipotetico etichettato come &quot;Gene # 6&quot;. Il pannello superiore illustra il timidina e il pannello inferiore della secrezione di insulina.

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Non ci sono conflitti di interesse dichiarati.

Stabilire percorsi che possono essere modulate per stimolare la replicazione e migliorare la funzione delle cellule beta sono importanti per entrambe le forme principali di diabete. Poiché funzionale massa di cellule beta dipende dalla esistenza e la funzione di cellule che secernono insulina, valutando questi determinanti simultaneamente ha i suoi vantaggi. Questo protocollo descrive un protocollo semplificato per identificare se la sovraespressione o la soppressione di una proteina porta a variazioni di massa funzionale delle cellule beta in vitro, che possono poi essere testati per l&#39;efficacia in vivo. Una limitazione di questo protocollo è che l&#39;isolotto è un micro-organo composto da molti tipi di cellule, inclusi ma non limitati a alpha, beta, delta, epsilon, e le cellule PP. Un cambiamento nella replicazione isolotto non può completamente traducono in un cambiamento nella replicazione delle cellule beta causa 80-90% delle cellule del isolotto ratto sono cellule beta. La possibilità che una variazione osservata in isolottola replica è dovuto alla non-beta replicazione cellulare esiste. Quindi, un passo logico e conferma accanto a questo protocollo potrebbe essere esaminando la replicazione delle cellule beta con l&#39;uso di analoghi della timidina accoppiati ad immunofluorescenza o analisi FACS 5,6. Questa analisi conferma può anche alleviare il possibile problema di non-specifica timidina nel isolotti indipendenti di proliferazione. Un altro inconveniente risiede nel potenziale efficienza di trasduzione di adenovirus. Una efficienza di trasduzione del 60-70% è ragionevole da raggiungere, ma un fattore determinante per l&#39;efficacia è la tempistica di trasduzione adenovirale. E &#39;essenziale per la cultura delle isole isolate nelle adenovirus il più presto possibile per massimizzare l&#39;efficienza di trasduzione. Entro poche ore dopo l&#39;isolamento isolotto l&#39;isolotto comincia a contrarsi, limitando così la capacità del adenovirus di penetrare in profondità nel cuore dell&#39;isolotto. L&#39;uso di un costrutto reporter, come ad esempiouno che esprime GFP adenovirus, accoppiata con la microscopia confocale può essere utile per valutare l&#39;efficienza di trasduzione. I vantaggi principali di questo protocollo sono: 1) l&#39;efficienza dei test determinanti multipli di massa funzionale delle cellule beta nella stesso pool di isolotti e 2) i numeri piccoli isolotti necessari per eseguire il protocollo (un tipico isolamento isolotto ratto produce 400 isolotti) . Questi vantaggi consentono questo protocollo per essere utilizzato come strumento di screening per i geni più ad un ritmo moderato.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> RPMI 1640 mezzi </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> Penicillina / streptomicina </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 6-pozzetti </td><td> BD-Falcon </td><td> 35-1146 </td><td> Non-TC trattati </td></tr><tr><td> [Metil-3 H]-timidina </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Micro-provette da centrifuga; </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> Soluzione 1 M </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-glucosio </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> Scintillazione tubo </td><td> Sarstedt </td><td> 58,536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Scintillazione tappo del tubo di conteggio </td><td> Sarstedt </td><td> 65,816 </td><td></td></tr><tr><td> Econo-Safe cocktail di conteggio </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> Insulina 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> Attrezzatura </td></tr><tr><td> Centrifuga </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Scintillazione portaprovette </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Contatore a scintillazione liquida </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

Omeostasi del glucosio è principalmente controllata dal sistema endocrino gli ormoni insulina e glucagone, secreto dal pancreas beta e le cellule alfa, rispettivamente. Functional massa di cellule beta è determinata dalla massa anatomica cellule beta così come la capacità delle cellule beta per rispondere a un carico di nutrienti. Una perdita di massa funzionale delle cellule beta è fondamentale per entrambe le forme principali di diabete 1-3. Considerando che i risultati in declino funzionale massa di cellule beta da un attacco autoimmune nel diabete tipo 1, diabete di tipo 2, questo decremento si sviluppa sia da una incapacità delle cellule beta di secernere insulina in modo appropriato e la distruzione delle cellule beta da una squadra di meccanismi. Pertanto, gli sforzi per ristabilire la massa funzionale delle cellule beta sono di primaria importanza al trattamento migliore e potenziali cure per il diabete. Gli sforzi sono in corso per identificare i meccanismi molecolari che possono essere sfruttate per stimolare la replicazione e migliorare la funzione delle cellule beta.Idealmente, gli obiettivi terapeutici potrebbe migliorare sia la crescita e la funzione delle cellule beta. Forse ancora più importante però è quello di identificare se una strategia che stimola la crescita delle cellule beta arriva al costo di compromettere la funzione delle cellule beta (come nel caso di alcuni oncogeni) e viceversa. Sistematicamente la soppressione o sovraespressione l&#39;espressione di geni bersaglio in isole isolate di ratto, si possono identificare potenziali bersagli terapeutici per aumentare la massa funzionale delle cellule beta 4-6. I vettori adenovirali possono essere impiegati per proteine ​​efficiente esprimono molti o smontabile in isole di ratto isolati 4,7-15. Qui, presentiamo un metodo per modificare l&#39;espressione genica utilizzando trasduzione adenovirale e valutare la replicazione e la funzione delle cellule insulari beta in isole di ratto isolati (Figura 1). Questo metodo è stato utilizzato in precedenza per identificare nuovi bersagli che modulano la replicazione delle cellule beta o una funzione 5,6,8,9,16,17.

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

Questo protocollo permette di identificare fattori che modulano funzionale massa delle cellule beta per trovare potenziali bersagli terapeutici per il trattamento del diabete. Il protocollo consiste in un metodo semplificato per valutare la replicazione delle isole e la funzione delle cellule beta in isole di ratto isolati dopo manipolazione genica con adenovirus.

Isolotti roditori Valutazione di replica e la funzione delle cellule beta in Adenovirally-trasdotte Isolati

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

Nanyang Technological University

Research

JoVE Journal

Medicine

13.9K Views.  Indiana University School of Medicine. Questo protocollo permette di identificare fattori che modulano funzionale massa delle cellule beta per trovare potenziali bersagli terapeutici per il trattamento del diabete. Il protocollo consiste in un metodo semplificato per valutare la replicazione delle isole e la funzione delle cellule beta in isole di ratto isolati dopo manipolazione genica con adenovirus.

Video: 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.

Valutazione della funzione cardiaca e Energetica nel Cuori mouse isolato mediante 31 P spettroscopia NMR

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

Ricerca

Medicina

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.

Valutazione della funzione endoteliale vasodilatatore con l&#39;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-meccanico Analisi dei isolata del muscolo scheletrico

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 ...

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.

Valutare i cambiamenti teratogeni in un modello di Zebrafish di esposizione alcolica fetale

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.

Protocolli di colorazione di isole pancreatiche umane

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

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 &#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.

A High-contenuti<em&gt; In Vitro</em&gt; La replica della piattaforma Discovery isole pancreatiche β-cellule

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'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; Modeling (q3DISM) di cerebrale amiloide-beta fagocitosi in roditori modelli di malattia di 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.

Nerve-sparing Mid-ostruzione uretrale (Nemo) nel femminile piccoli roditori

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

Intraportal Transplantation of Pancreatic Islets in Mouse Model

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

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

Intraportal trapianto di isole pancreatiche nel modello del topo

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

A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a computerized functional skills training program that was paired in half of the participants with a commercially available cognitive training (CCT) program.
Non-impaired older individuals (NC) aged 60+ (n=45) and similarly aged individuals with mild cognitive impairment (MCI; n=50) were randomized to receive 12 weeks of twice-weekly computerized functional skills training (CFST) or 12 weeks of twice-weekly sessions split between CCT and CFST. Skills trained were use of an ATM; internet banking; ticket kiosk; telephone and internet prescription refill; medication management; and internet shopping. As with previous functional capacity assessments, we focus on completion time for each simulation.
51 participants completed the training program, either by mastering all 6 tasks (34) or completing 12 weeks of training. 44 more participants completed 4 or more training sessions so they were also analyzed for improvement up to their last training session. Completion time for all 6 tests significantly improved from the baseline assessment to the final training session in both groups of participants (all p&#60;0.001 with an average improvement in task completion time of 45%). Further, there was no differential improvement in MCI and NC in the 6 tests from baseline to end of training (all t&#60;1.66, all p&#62;0.12). Finally, combined CCT plus CFST did not differ from CSFT alone on any of the percent-change score measures (all t&#60;1.64, all p&#62;0.11).
Both NC and MCI groups evidenced substantial improvements in performance. CCT supplementation led to similar functional gains with half as many training sessions. The NC participants proceeded through the training fairly rapidly even without CCT supplementation; MCI participants required more training but learned equivalently. These findings suggest that even in cases with memory impairments, functional skills can be efficiently learned with training.

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.

Un programma computerizzato di valutazione e formazione delle competenze funzionali, destinato alle competenze funzionali quotidiane basate sulla tecnologia