Este trabalho foi apoiado pela concessão DK078732 do NIH (a FPT).

1. Transdução adenoviral e cultura de ilhotas de ratos <ol><li> Prepara-se uma placa de 6 poços de cultura de tecido não-revestido por adição de 2 ml de meio (RPMI 1640 contendo 8 mM de glucose, 10% de soro fetal bovino, 50 unidades / ml de penicilina, e 50 ug / ml de estreptomicina) para o número necessário de poços. Por exemplo, uma experiência típica pode exigir três poços - um para cada um controlo não-vírus, um controle de vírus (por exemplo, GFP-expressando adenovírus), eo grupo experimental. </li><li> Aqueceu-se a placa a 37 ° C, colocando-a em uma cultura de tecidos incubadora durante pelo menos 30 min. </li><li> Imediatamente após o isolamento de rato ilhota 18,19, o local de 100-200 ilhotas em poços individuais da placa de cultura de 6 poços tecido não-revestido. Sessenta ilhotas são necessários para a secreção de insulina e ensaios de incorporação de timidina. As ilhotas restantes podem ser utilizados para o isolamento de RNA para estudos de expressão génica ou isolamento de proteínas para immunoblotting. </li></ol> [Nota: A partir deste ponto em diante, siga protocolos institucionais para o manejo, uso e descarte de materiais perigosos.] <ol start="4"><li> Agite ligeiramente a placa para trazer as ilhotas para o centro do poço. </li><li> Pipetar o adenovírus directamente sobre as ilhotas no centro do prato. Use 100-500 multiplicidades de infecção (MOI, a proporção de células alvo para virais unidades formadoras de placas). </li><li> Deixe o resto ilhotas durante 5 min. </li><li> Colocar a placa na incubadora de cultura de tecido (37 ° C, 5% de CO 2). </li><li> Após 24 h, agite a placa para trazer as ilhotas para os centros dos poços e transferir as ilhotas utilizando uma micropipeta P200 para uma nova poços contendo meio fresco. Se as ilhotas tornar-se ligado à placa, eles podem ser desalojado suavemente com a ponta da pipeta. </li></ol> [Nota: Para verificar eficiên transdução adequadacy, a utilização de um vírus de controlo expressando GFP é benéfico, tal como ilhotas pode então ser trabalhada por meio de microscopia confocal para verificar a penetração do adenovírus no núcleo das ilhotas.] <ol start="9"><li> Cultura das ilhotas para uma h 24-72 adicional, dependendo da temporização desejada do experimento a partir de estudos piloto de optimização. Por exemplo, a indução de uma resposta proliferativa podem exigir tempos que variam de 24-72 h ou knockdown do gene de interesse pode exigir 48 ou 72 horas. Transfira as ilhotas de mídia fresco todos os dias. </li><li> Para a 24 h final da experiência, a cultura das ilhotas em meios contendo 1 uCi [metil-3 H] -thymidine/ml meios de comunicação (geralmente 1 uL de timidina / ml a media). </li></ol> [Nota: A partir deste ponto em diante, siga protocolos institucionais para o manejo, uso e descarte de materiais radioativos.] 2. Teste de secreção de insulina <ol><li> Preparar otampão de secreção de ensaio (SAB) de solução stock de 10X (1,14 M de NaCl, 47 mM de KCl, 12 mM de KH 2 PO 4, 11,6 mM MgSO4) e CaCl 2 100X solução stock (0,25 M de CaCl2). Estas soluções de reserva pode ser preparado antecipadamente e guardado à temperatura ambiente. </li><li> Recém-prepará 50 ml de ORS de trabalho (5 mL de SAB 10X, 1 ml de HEPES 1 M, 0,5 ml de 100X de CaCl2, 0,28 ml de BSA 35%, 0,11 g de NaHCO3, e água estéril para 50 ml) num 50-ml cónica do tubo e aquecido a 37 ° C, colocando em um banho-maria a 37 ° C. </li><li> Pipetar 10 ml da ORS de trabalho para um tubo de 15 ml e adicionar 66,8 ul de 2,5 M D-glucose para preparar o glucose elevada (16,7 mM) SAB. </li><li> Adicionar 44,8 ul de 2,5 M D-glucose ao ml restantes 40 da ORS de trabalho para preparar a glicose baixa (2,8 mM) SAB. </li><li> Rotular três tubos de 1,7 ml de microcentrífuga para cada poço da placa de 6 poços e adicionar 1 ml de salino tamponado com fosfato (PBS). </li></ol&gt; [Nota: Como as ilhotas são radioativos, siga protocolos institucionais para o manejo, uso e descarte de materiais radioativos.] <ol start="6"><li> Colocar 20 ilhotas em cada tubo de microcentrífuga. Fazer com que cada tentativa de adicionar ilhotas de tamanho comparável a cada tubo de microcentrífuga. Por exemplo, cada tubo pode conter 5 a médio, 10-, pequenas e 5 de grande porte ilhotas (ver Figura 1). </li></ol> [Nota: Islets pode ser visualizada usando um estereoscópio dissecando ou um microscópio padrão.] <ol start="7"><li> Após as ilhotas se estabeleceram na parte inferior do tubo por gravidade (~ 2 min), aspirar o PBS com uma micropipeta e descartar. </li></ol> [Nota: Como uma alternativa para a sedimentação por gravidade, os tubos podem ser centrifugado a 300 xg durante 1 min.] <ol start="8"><li> Para a pré-incubação, adicionar 400 uL da glu baixocose SAB, colocar os tubos (com as suas tampas abertas) para o tecido incubador de cultura (37 ° C, 5% de CO 2), e pré-incubar durante 60 min. Aspirar a pré-incubação baixa glicose SAB e descarte. </li><li> Para a secreção de insulina basal, adicionar 400 uL da baixa glicose SAB, colocar os tubos (com as suas tampas abertas) para o tecido incubador de cultura (37 ° C, 5% de CO 2), e incubar durante 60 min. Recolher o baixo de glicose SAB e poupar para o radioimunoensaio de insulina. </li><li> Para a secreção de insulina estimulada, adicionar 400 uL da alta glucose SAB, colocar os tubos (com as suas tampas abertas) para o tecido incubador de cultura (37 ° C, 5% de CO 2), e incubar durante 60 min. Recolher o alto de glicose SAB e poupar para o radioimunoensaio de insulina. </li></ol> 3. Ensaio de Incorporação de timidina <ol><li> Adicionar 1 ml de PBS; após as ilhotas ter assente no fundo do tubo, por gravidade, aspirar o PBS com uma micropipeta, descartar, e repetir estepasso uma vez. </li><li> Adicionar 500 uL de gelo de ácido tricloroacético frio (TCA, 10% w / v) e incubar em gelo durante 30 min. </li><li> Centrifugar os tubos a 16 000 xg durante 3 min a 4 ° C. </li><li> Aspirar a ATC, adicionar 80 ul de 0,3 N NaOH, e incubar durante 30 min à temperatura ambiente. Durante este tempo, vigorosamente vórtice as amostras para 5-10 s cada 10 min. </li><li> Adicionar 4 ml de cocktail Econo-segura contagem para 7 ml tubos líquidos contagem de cintilação. </li><li> Adicionar 50 ul da amostra para o tubo de contagem de cintilação, limitar o tubo, agitar brevemente, e contar num contador de cintilação líquida. </li><li> Medir a concentração de proteína utilizando o ácido bicinconínico (BCA) de ensaio e 10 ul de amostra de acordo com o protocolo do fabricante. </li></ol> 4. Análise de Dados <ol><li> Realizar o radioimunoensaio insulina seguindo o protocolo do fabricante. </li><li> Normalizar a secreção de insulina e os dados de incorporação de timidina com a proteína concentration. </li></ol> 5. Os resultados representativos Um exemplo da experiência para avaliar a replicação de ilhotas e função das células beta em ilhotas de ratos é mostrado na Figura 2. Este exemplo mostra que a superexpressão adenoviral de &quot;Gene # 6&quot; hipotético robustamente estimula a replicação ilhota sem alterar a função das células beta. No painel superior, os resultados do ensaio de incorporação de timidina demonstrar que o aumento da expressão de &quot;Gene # 6&quot; aumenta a síntese de DNA, tal como medido pela incorporação de timidina. Como a maioria das células no ilhéu do rato são células beta, é provável que este aumento na incorporação de timidina indica um aumento da replicação das células beta. No entanto, as experiências de confirmação deve ser realizado para estabelecer firmemente este. No painel de fundo, os resultados do ensaio de secreção de insulina demonstrar que a superexpressão da &quot;Gene # 6&quot; não alterou uma das funções celulares primárias beta, isto é, insulin secreção de glicose baixa e alta. A qualidade do isolamento dos ilhéus e saúde dos ilhéus após o tratamento com adenovírus é indicado pelo aumento vezes na secreção de insulina em concentrações de glicose de baixa e alta. Se o aumento da expressão da função &quot;Gene # 6&quot; célula deficiente beta, este seria provavelmente como um decréscimo na insulina segregada em concentrações elevadas, estimuladores de glicose (16,7 mM). Uma curva dose-resposta para várias concentrações de glicose pode também ser realizada. <img src="/files/ftp_upload/4080/4080fig1.jpg" alt="A Figura 1"/> Figura 1. Geral de protocolo para avaliar a replicação de ilhotas e função das células beta em ilhéus isolados de ratos após adenoviral mediadas alterações na expressão do gene. Ilhotas de ratos recém-isoladas são expostos a adenovírus durante 24 horas e então cultivadas até 96 h. Incorporação de timidina são avaliados no 24 h final, seguido pela medição da secreção de insulina noglicose baixa e alta. <img src="/files/ftp_upload/4080/4080fig2.jpg" alt="A Figura 2"/> Figura 2. Os resultados de um experimento utilizando um adenovírus e controle de um adenovírus com superexpressão de um gene hipotético rotulados como &quot;Gene # 6&quot;. O painel superior mostra a incorporação de timidina e do painel de fundo a secreção de insulina.

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Vias, que estabelece que pode ser modulada para estimular a replicação e melhorar a função das células beta são relevantes para ambas as formas principais de diabetes. Devido massa celular funcional beta é dependente da existência e função de células secretoras de insulina, avaliando estes determinantes simultaneamente tem as suas vantagens. Este protocolo descreve um protocolo simplificado para identificar se a superexpressão ou supressão de uma proteína leva a alterações na massa funcional das células...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> RPMI 1640 meios </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> A penicilina / estreptomicina </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 6-bem placa </td><td> BD-Falcon </td><td> 35-1146 </td><td> Non-TC tratada </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-centrífuga tubos </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> MgSO4 </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> Solução 1 M </td></tr><tr><td> 35% de BSA </td><td> Sigma </td><td> A7979 </td><td></td></tr><tr><td> De NaHCO3 </td><td> Acros </td><td> 42427 </td><td></td></tr><tr><td> d-glicose </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> Contagem de cintilação tubo </td><td> Sarstedt </td><td> 58,536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Cintilação tampa do tubo de contagem </td><td> Sarstedt </td><td> 65,816 </td><td></td></tr><tr><td> Econo-Safe cocktail contando </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> RIA Insulina </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> Equipamento </td></tr><tr><td> Centrifugar </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Contagem de cintilação suporte de tubos </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Contador de cintilação líquida </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

Homeostase da glucose é principalmente controlada pela insulina hormonas endócrinas e glucagon, secretada a partir da beta pancreáticas e células alfa, respectivamente. Massa celular funcional beta é determinado pela massa anatómica beta celular, bem como a capacidade das células beta para responder a uma carga de nutriente. Uma perda de massa funcional das células beta é central para ambas as formas principais de diabetes 1-3. Considerando que as declínio da capacidade funcional das células beta resultados de massa de um ataque auto-imune em pacientes com diabetes tipo 1, diabetes tipo 2, este decréscimo se desenvolve tanto uma incapacidade das células beta a secretar insulina de forma apropriada e destruição das células beta de um quadro de mecanismos. Assim, os esforços para restabelecer a massa de células beta funcional são fundamentais para o melhor tratamento e possíveis curas para o diabetes. Esforços estão em andamento para identificar caminhos moleculares que podem ser exploradas para estimular a replicação e melhorar a função das células beta.Idealmente, alvos terapêuticos iria melhorar tanto o crescimento das células beta e função. Talvez o mais importante é que para identificar se uma estratégia que estimula o crescimento das células beta está no custo da função das células beta prejudicando (tal como com alguns oncogenes) e vice-versa. Sistematicamente suprimindo ou superexpressão de expressão de genes-alvo em ilhéus isolados de ratos, é possível identificar potenciais alvos terapêuticos para o aumento da massa funcional das células beta 4-6. Vectores adenovirais podem ser empregues para proteínas eficientemente superexpressam ou knockdown em ilhéus isolados de ratos 4,7-15. Aqui, nós apresentamos um método para manipular a expressão do gene utilizando transdução adenoviral e avaliar a replicação de ilhotas e função das células beta em ilhéus isolados de ratos (Figura 1). Este método tem sido utilizado anteriormente para identificar novos alvos que modulam a replicação das células beta ou função 5,6,8,9,16,17.

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

Este protocolo permite uma para identificar factores que modulam a massa de células funcional de beta para encontrar potenciais alvos terapêuticos para o tratamento da diabetes. O protocolo é constituído por um método simplificado para avaliar a replicação de ilhotas e função das células beta em ilhéus isolados de ratos após a manipulação da expressão do gene com adenovírus.

Avaliação de replicação e função das células beta no Adenovirally transduzidas isoladas ilhotas de roedores

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

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13.9K visualizações.  Indiana University School of Medicine. Este protocolo permite uma para identificar factores que modulam a massa de células funcional de beta para encontrar potenciais alvos terapêuticos para o tratamento da diabetes. O protocolo é constituído por um método simplificado para avaliar a replicação de ilhotas e função das células beta em ilhéus isolados de ratos após a manipulação da expressão do gene com adenovírus.

Vídeo: 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.

Avaliação da função cardíaca e Energetics em Corações Isolados rato Usando 31 P NMR Spectroscopy

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

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.

Avaliação da função endotelial com a Vasodilatador 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-mecânico Análise do músculo esquelético isolado

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.

Isolamento de Ilhotas Humanos de pacientes parcialmente pancreatectomizado

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.

Avaliar as alterações teratogênicos em um modelo de Zebrafish de exposição fetal ao álcool

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.

Protocolos de coloração de ilhotas pancreáticas humanas

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; Mouse Modelo de Diabetes Tipo 2: Isolamento de ilhotas pancreáticas e células vivas 2-Photon Imagens de ilhotas intactas

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.

A-alta conteúdo<em&gt; In Vitro</em&gt; Replicação plataforma de descoberta de células-β das ilhotas pancreáticas

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 quantitativa<em&gt; In Silico</em&gt; Modelagem (q3DISM) de beta-amilóide cerebral fagocitose em modelos de roedores da doença de 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.

-Nervo poupadores Mid-obstrução uretral (Nemo) em fêmeas pequenos roedores

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