Bu çalışma NIH (PTF kadar) hibe DK078732 tarafından desteklenmiştir.

1. Sıçan Adacıklar ve adenoviral İletimi ve Kültürleme <ol><li> Gerekli sayıda ortam, 2 ml (8 mM glikoz,% 10 fetal bovin serumu, 50 ünite / ml penisilin ve 50 ug / ml streptomisin içeren RPMI 1640 ortamı) ilave edilerek bir 6-sıra non-doku kültürü plakası kaplamalı hazırlamak kuyu. No-virüsü kontrolü için tek her biri, bir virüs kontrolü (örn., GFP-eksprese adenovirüs) ve deney grubu - Örneğin, tipik bir deneyde, üç kuyu gerektirebilir. </li><li> En azından 30 dakika için bir doku kültürü inkübatöründe yerleştirilerek 37 ° C ye ısınmasına plakası. </li><li> Hemen 6-sıra dışı doku kültürü kaplı plaka bireysel kuyulara sıçan adacık izolasyonu 18,19, yerde 100-200 adacıklar takip. Altmış adacıklar insülin salgılanması ve timidin dahil testleri için gereklidir. Kalan adacıklar gen ekspresyonu ya immunoblotting çalışmaları için protein izolasyon için RNA izolasyon için kullanılabilir. </li></ol> [Not: Bu noktadan itibaren, biyolojik tehlikeli malzeme taşıma, kullanma, ve bertaraf için kurumsal protokolleri takip edin.] <ol start="4"><li> Yavaşça girdap de merkezine adacıklar getirmek için plaka. </li><li> Doğrudan çanağı merkezinde adacıklar üzerine adenovirüs Pipeti. Enfeksiyon 100-500 çokluklar (İçişleri Bakanlığı, viral plak oluşturan birim için hedef hücrelerin oranı) kullanın. </li><li> 5 dk için adacıklar dinlenmeye bırakılır. </li><li> Doku kültürü inkübatöründe (37 ° C&#39;de,% 5 CO2) in plaka yerleştirin. </li><li> 24 saat sonra yavaşça girdap kuyuların merkezlerine adacıklar getirmek ve iyi taze medya içeren yeni bir P200 mikropipet kullanarak adacıklar aktarmak için plaka. Adacıklar plakasına bağlı olursanız, onlar yavaşça pipet ucu ile yerinden edilebilir. </li></ol> [Not: Yeterli transdüksiyon Verim doğrulamak içinadacıklar sonra adacık çekirdek içine adenovirüs penetrasyon doğrulamak için konfokal mikroskopi yansıması gibi cy, bir kontrol virüs ifade GFP kullanılması yararlı olur.] <ol start="9"><li> Optimizasyon pilot çalışma gelen deney istenen zamanlama bağlı olarak ek 24-72 saat boyunca kültürün adacıklar,. Örneğin, bir çoğalma tepkisi indüksiyonu 24-72 arası bir şekilde H veya ilgi genindeki demonte 48 ya da 72 saat arasında değişen gerektirebilir kez gerektirebilir. Taze medya her gün adacıklar aktarın. </li><li> Deney, 1 içeren ortam içinde kültüre adacıklar nihai 24 saat süreyle μCi [metil-3 H] -thymidine/ml ortam (genellikle 1 ul timidin / ml ortamı). </li></ol> [Not: Bu noktadan itibaren, radyoaktif maddelerin taşınması, kullanımı ve bertaraf edilmesi için kurumsal protokolleri takip edin.] 2. İnsülin salgılanması Assay <ol><li> Hazırlayınsalgılanması deney tamponu (SAB) 10X stok çözeltisi (1.14 M NaCl, 47 mM KCI, 12 mM KH 2 PO 4, 11.6 mM MgSO 4) ve CaCI2 100X stok çözeltisi (0.25 M CaCI2). Bu stok çözümler önceden hazırlanan ve oda sıcaklığında saklanabilir. </li><li> Taze olarak çalışan SAB (10X SAB, 1 M HEPES, 100X CaCI2 ve 0.5 ml,% 35 BSA, 0.11 g NaHCO 3 0.28 ml 1 ml, ve steril su ile 50 ml 5 mL) içinde 50 ml hazırlanması 50 ml tüp konik ve ılık 37 ° C 37 ° C su banyosunda koyarak. </li><li> Pipet 10 15 ml konik tüp içine çalışma SAB ml ve yüksek glukoz (16.7 mM) SAB hazırlamak için 2.5 M D-glikoz 66.8 ul ekleyin. </li><li> Düşük glukoz (2.8 mM) SAB hazırlamak için çalışma SAB kalan 40 ml 2.5 M D-glikoz 44.8 ul ekleyin. </li><li> Etiket üç 1.7 ml mikrosantrifüj 6-kuyulu plakanın her bir kuyucuk için tüpler ve fosfat-tamponlu salin (PBS) 1 ml ilave edilir. </li></ol&gt; [Not: adacıklar radyoaktif olduğundan, radyoaktif maddelerin taşınması, kullanımı ve bertaraf edilmesi için kurumsal protokolleri takip edin.] <ol start="6"><li> Her mikrosantrifüj tüpüne 20 adacıktan yerleştirin. Her mikrosantrifüj tüpüne karşılaştırılabilir büyüklükte adacıklar eklemek için her türlü girişimi olun. Örneğin, her bir tüp 5 küçük, 10 orta ve 5 büyük ölçekli adacıklar (bakınız Şekil 1) ihtiva edebilir. </li></ol> [Not: Islets bir diseksiyon stereoskop veya standart bir mikroskop kullanılarak görüntülenebilir.] <ol start="7"><li> Adacıklar ağırlık (~ 2 dk) göre tüp alt yapıldıktan sonra, mikropipet ile PBS aspire ve atılır. </li></ol> [Not: yerçekimi ile çökeltme alternatif olarak, boru 1 dakika boyunca 300 x g&#39;de santrifüje tabi olabilir.] <ol start="8"><li> Ön kuluçka için, düşük Glu 400 ul ilavecose SAB, doku kültüründe inkübatöründe (37 ° C&#39;de,% 5 CO2), ve 60 dakika boyunca ön-kuluçkaya yatmaktadır içine boru (bunların kapaklar açık olan) yerleştirin. Ön inkübasyon düşük glikoz SAB ve atın aspire. </li><li> Temel insülin salgılanmasını için, düşük glikoz SAB 400 ul ilave, doku kültüründe inkübatöründe (37 ° C&#39;de,% 5 CO2) içine boru (bunların kapaklar açık olan) yerleştirmek, ve 60 dakika süreyle inkübe edilir. Düşük glukoz SAB toplayın ve insülin radioimmunoassay için kaydetmek. </li><li> Uyarılmış insülin salgılanmasını için, yüksek glikoz SAB 400 ul ilave, doku kültüründe inkübatöründe (37 ° C&#39;de,% 5 CO2) içine boru (bunların kapaklar açık olan) yerleştirmek, ve 60 dakika süreyle inkübe edilir. Yüksek glukoz SAB toplayın ve insülin radioimmunoassay için kaydetmek. </li></ol> 3. Timidin Ortaklığın Assay <ol><li> 1 ml PBS Add; adacıklar yerçekimi tarafından borusunun alt yapıldıktan sonra, mikropipet ile PBS aspire atmak ve bu, tekrarbir kez adım. </li><li> 500 ul buz gibi soğuk trikloroasetik asit (TCA,% 10 w / v) eklenir ve 30 dakika süreyle buz üzerinde inkübe edilir. </li><li> 4 3 dakika ° C için 16 000 xg&#39;de tüpler santrifüje </li><li> TCA aspire, 0.3 N NaOH ile 80 ul ilave edildi ve oda sıcaklığında 30 dakika inkübe edilir. Bu süre esnasında, 5-10 s boyunca kuvvetli bir şekilde vorteks numuneleri, her 10 dak. </li><li> 7 ml sıvı sintilasyon sayım tüplerine Econo-güvenli sayma kokteyli 4 ml ekleyin. </li><li> Sintilasyon sayacı tüpüne numunenin 50 ul ekleme, tüp kap kısa bir süre için çalkalanır, ve bir sıvı sintilasyon sayacı içinde sayılır. </li><li> Üreticinin protokolüne göre bicinchoninic asit (BCA) test, örnek 10 ul kullanılarak protein konsantrasyonu ölçülür. </li></ol> 4. Veri Analizi <ol><li> Üreticinin protokolü takip insülin radioimmunoassay gerçekleştirin. </li><li> Protein kons ile insülin salgılanması ve timidin dahil veri Normaleentration. </li></ol> 5.. Temsilcisi Sonuçlar Sıçan adacıklarında adacık replikasyonu ve beta hücresi fonksiyonunu değerlendirmek için deney için bir örnek, Şekil 2&#39;de gösterilmiştir. Bu örnek, sağlam beta hücre fonksiyonu değiştirmeden adacık çoğaltma uyarır varsayımsal &quot;# 6 Gen&quot; bu adenoviral aşırı gösterir. Üst panosunda, timidin dahil testte sonuçlar &quot;Gen # 6&quot; ifadesini arttırarak timidin eklenmesi ile ölçülen DNA sentezi, artırdığını göstermektedir. Sıçan adacık hücrelerin en beta hücrelerinin olduğundan, timidin dahil bu artış beta hücre replikasyon bir artış gösterir olasıdır. Bununla birlikte, doğrulayıcı deneyler sıkıca Bu kurmak için gerçekleştirilmesi gerekir. Alt panelinde, insülin salgılanması testte sonuçlar &quot;Gen # 6&quot; i, yani birincil beta hücre fonksiyonlarının bir değişiklik olmadı bu aşırı göstermekdüşük ve yüksek glukoz de nsulin salgısı. Adenovirüsler ile tedaviyi takiben adacık adacık izolasyonu ve sağlık kalitesi düşük ve yüksek glukoz konsantrasyonlarında insülin salınımını kat artış ile gösterilir. &quot;Gen # 6&quot; bozulmuş beta hücre fonksiyon ifadesini arttırarak, bu olasılığı yüksek, uyarıcı glukoz konsantrasyonları (16.7 mM) olarak salgılanan insülin bir azalma olarak yansıtılacaktır. Glukoz biyokatalist için bir doz-yanıt eğrisi de yapılabilir. <img src="/files/ftp_upload/4080/4080fig1.jpg" alt="Şekil 1"/> Şekil 1. Gen ekspresyonu adenoviral aracılı değişiklikler sonrasında izole sıçan adacıklarında adacık çoğaltma ve beta hücre fonksiyonu değerlendirmek için protokol bakış. Taze izole sıçan adacıklar 24 saat adenovirüsler maruz kalan ve daha sonra 96 ​​saat kadar kültürlenmiştir. Timidin dahil olarak insülin salgılanmasını ölçülmesi ile takip, nihai 24 saat içinde değerlendirilirdüşük ve yüksek glukoz. <img src="/files/ftp_upload/4080/4080fig2.jpg" alt="Şekil 2"/> Şekil 2. Bir kontrol adenovirüs ve adenovirüs aşırı &quot;Gen # 6&quot; olarak adlandırılan varsayımsal bir gen üzerinde yapılan bir deneyde elde edilen sonuçlar. Üst panel timidin dahil olması ve alt panelini insülin salgılanmasını gösterir.

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Çıkar çatışması ilan etti.

Çoğaltma teşvik ve beta hücrelerinin işlevini geliştirmek için modüle olabilir kurulması yollarının her iki diyabet büyük formlar alakalı. Işlevsel beta hücre kitlesi bu belirleyicileri değerlendirmek, insülin salgılayan hücrelerin varlığı ve fonksiyonu bağlı olduğundan aynı anda avantajları vardır. Bu protokolün bir proteini aşırı ekspresyonu ya da bastırma sonra in vivo etki için test edilebilir in vitro olarak fonksiyonel beta hücre kütlesi değişikliklere yol a...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> RPMI 1640 ortamı </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> Penisilin / streptomisin </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 6-iyi plaka </td><td> BD-Falcon </td><td> 35-1146 </td><td> Sigara TC tedavi </td></tr><tr><td> [Metil-3 H] timidin- </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Mikro-santrifüj tüpü </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> CaCI2 </td><td> Acros </td><td> 34961 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H0887 </td><td> 1 M solüsyon </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-glukoz </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> Sintilasyon tüp sayma </td><td> Sarstedt </td><td> 58.536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Sintilasyon sayma tüp kap </td><td> Sarstedt </td><td> 65.816 </td><td></td></tr><tr><td> Econo-Güvenli sayma kokteyl </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> İnsülin RIA </td><td> Siemens </td><td> TKIN2 </td><td></td></tr><tr><td> BCA Test Kiti </td><td> Thermo Scientific </td><td> 23250 </td><td></td></tr><tr><td></td><td></td><td></td><td> Ekipman </td></tr><tr><td> Santrifüj </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Sintilasyon tüp raf sayma </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Sıvı sintilasyon sayacı </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

Glikoz homeostazı, öncelikle, sırasıyla, pankreas beta ve alfa hücrelerden salgılanan, endokrin hormonlar insülin ve glukagon tarafından kontrol edilir. Fonksiyonel beta hücre kütlesi anatomik beta hücre kütlesi gibi bir besin yük yanıt beta hücrelerinin yeteneği ile belirlenir. Işlevsel beta hücre kitlesi kaybı diyabet 1-3 iki büyük formlar merkezidir. Tip 1 diyabetin otoimmün bir saldırı azalan işlevsel beta hücre kitlesi sonuçları ise, tip 2 diyabet, bu eksiltme uygun insülin salgılamaya beta hücrelerinin bir yetersizlik ve mekanizmalar bir kadro beta hücrelerinin yok hem de gelişir. Böylece, işlevsel beta hücre kitlesi geri çabaları daha iyi tedavi ve diyabet için potansiyel tedavileri için her şeyden vardır. Çabaları çoğaltma teşvik ve beta hücrelerinin işlevini geliştirmek için kullanılabilir moleküler yolları belirlemek için çalışmalar devam etmektedir.İdeal olarak, tedavi hedefleri beta hücre büyümesi ve fonksiyonu hem de artıracak. Ancak belki de daha önemli beta hücre büyümesini uyarır bir strateji zedeleyen beta hücre fonksiyonu (bazı onkogenler olduğu gibi) ve tam tersi pahasına geliyor olmadığını belirlemektir. Izole sıçan adacıklar hedef genlerin sistematik baskılayarak veya aşırı ifadesi olarak, bir artan işlevsel beta hücre kitlesi 4-6 için potansiyel tedavi hedefleri belirleyebilir. Adenoviral vektörler izole sıçan adacıklar 4,7-15 verimli olarak artmış veya demonte proteinlerine istihdam edilebilir. Burada, adenoviral transdüksiyon kullanarak gen ekspresyon işlemek ve izole sıçan adacıklar (Şekil 1) adacık çoğaltma ve beta hücre fonksiyonu değerlendirmek için bir yöntem sunuyoruz. Bu yöntem, beta hücre çoğaltma veya fonksiyon 5,6,8,9,16,17 modüle yeni hedefler belirlemek için daha önce kullanılmıştır.

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

Bu protokol bir diyabet tedavisi için terapötik hedefleri bulmak için işlevsel beta hücre kitlesi modüle faktörleri belirlemek sağlar. Protokolü adenovirüsler ile gen ekspresyon manipülasyonu takiben izole sıçan adacıklarında adacık çoğaltma ve beta hücre fonksiyonu değerlendirmek için modern bir yöntem oluşur.

Çoğaltma ve Beta Hücre Fonksiyonu değerlendirilmesi Adenovirally-transdük İzole Kemirgen Islets

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 Görüntüleme.  Indiana University School of Medicine. Bu protokol bir diyabet tedavisi için terapötik hedefleri bulmak için işlevsel beta hücre kitlesi modüle faktörleri belirlemek sağlar. Protokolü adenovirüsler ile gen ekspresyon manipülasyonu takiben izole sıçan adacıklarında adacık çoğaltma ve beta hücre fonksiyonu değerlendirmek için modern bir yöntem oluşur.

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.

Kalp Fonksiyonu ve Enerji Kullanımı İzole Fare Kalpler Değerlendirilmesi 31 P NMR Spektroskopisi

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

Tıp

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.

Endo-PAT 2000 ile Endotel Vazodilatör Fonksiyon değerlendirilmesi

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.

İzole İskelet Kası Myo-mekanik Analizi

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.

Kısmen Pancreatectomized hastalar İnsan Islets izolasyonu

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

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.

İnsan Pankreas Adacıklar için boyama Protokoller

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; Tip 2 Diyabet Fare Modeli: Bozulmamış Islets Of Pankreas Adacık İzolasyonu ve Canlı hücre 2-Foton Görüntüleme

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.

Bir Yüksek içerik<em&gt; İn Vitro</em&gt; Pankreas Adacık β-hücre çoğaltma Keşif Platformu

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.

kantitatif 3D<em&gt; Siliko</emAlzheimer Hastalığının Kemirgen Modelleri Serebral Amiloid-beta Fagositoz&gt; Modelleme (q3DISM)

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.

Kadın Küçük Kemirgenler Sinir koruyucu Orta üretral Tıkanıklığı (Nemo)

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.

İntraportal fare modeli pankreas adacıkları nakli

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