The authors acknowledge all colleagues who made this work possible; Ann Demarr&#233;, Veerle Laurysens, Jan De Jonge and Erik Quartier for technical assistance. Financial support was from the VUB Research Council, the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), the Beta Cell Biology Consortium (BCBC), the Fund for Scientific Research Flanders (FWO), Diabetes Onderzoek Nederland (DON) and Interuniversity Attraction Pole (IAP).

Tüm manipülasyonlar Deneysel ve diğer bilimsel Amaçlı kullanılan Omurgalı Hayvanların Korunmasına İlişkin Avrupa Sözleşmesi tarafından verilen yönergeleri izleyin (ETS 123 ve ve 2010/63 / AB). Çalışma Alanının 1. Hazırlanması <ol><li> Özel bir hazırlık alanı, cerrahi alanı ve kurtarma alanında sağlayın. </li><li> Çevre kirletici maddelerin en aza indirmek için, bir düzgün akış kabininde Cerrahi işlemin tamamı yürütün. </li><li> Hazırlık, cerrahi ve kurtarma alanında ihtiyaç duyulan (Gereç ve Yöntem listelendiği gibi) uygun aseptik teknik kullanılarak, sarf malzemeleri birleştirin. </li><li> Cerrahi aletler cerrahi öncesi otoklavlanırlar emin olun. </li><li> Ameliyat sırasında sıcaklık dengelenmesi için 38 ° C&#39;lik bir sıcaklıkta bir çevrimli su ısıtma pedi sağlar. Steril su geçirmez ped ile ısıtma yastığı örtün. </li><li> En azından 6.3X bir büyütme ile bir işletim mikroskop sağlayın. </li><li> Bir enstrüman Steri kullanınBöyle sıcak bir boncuk sterilizatör olarak lizer, cerrahi işlemler arasında aletleri sterilize etmek. </li><li> Düz kağıt yatak ile kaplı geniş bir kafes oluşan bir kurtarma alanı hazırlayın </li></ol> 2. Cerrahi Hayvanlar hazırlanması <ol><li> PDL ve sham için, 8 haftalık erkek fareler standart kafesler içine yerleştirilir ve muhafaza edilen, 12 saat ışık / 12 saat karanlık çevrimi ve standart kemirgen diyeti ile ad libitum kullanımı. Not: Burada, BALB / cJrJ fareleri kullanabilir ama biz de başarılı bir şekilde diğer suşları ve çeşitli transgenik suşları kullanılmıştır. </li><li> (- 0.1 mg / kg 0.05) ameliyat öncesi 30 dakika önleyici analjezi gibi Buprenophine kullanın. </li><li> 10 mg / ksilazin kg - ketamin 100 mg / kg ve 5 intraperitoneal enjeksiyonu ile farelere anestezi. </li><li> Istemli hareket ve kas gevşeme kademeli kaybı gözlemleyerek doğru anesthetization değerlendirin. Ayak sıkıştırarak reflekslerin kaybı test edin. </li><li> Önceki oftalmik merhem sürüngözlerin ent kuruluk anestezi altında iken. </li></ol> 3. Cerrahi Sitesi Hazırlama <ol><li> Antiseptik Klorheksidin çözümü ile toraks ve karın dezenfekte edin. </li><li> 2,5 cm karın x 1.5 cm lik bir alanı tıraş. </li><li> Daha sonra klorheksidin çözeltisi, alkol çözeltisi ve klorheksidin solüsyonunun, nihai uygulama ile ıslatılmış gazlı bez tıraş alanı dezenfekte. </li><li> Hazırlanan cerrahi alan yukarı cerrahı bakacak şekilde cerrahi alanda hayvan yerleştirin. </li><li> Vücudun geri kalanı kaplayan steril çalışma alanı oluşturmak için ise maruz dezenfekte karın bölgesini terk bir açıklığı olan bir su geçirmez cerrahi örtü kullanarak fare örtün. Anestezi derinliği prosedürü öncesinde fare izleyin. </li></ol> 4. Pankreas Kanal Ligasyonu <ol><li> Laparotomi <ol><li> Steril kullanarak göbek için kılıç şeklinde bir süreç uzanan deri bir üst orta hat kesi yapmakCerrahi bıçak. Altta yatan linea alba ve üst karın kadranı ortaya koymak için steril makas kullanılarak Periton ayırın. </li><li> Steril% 0.9 sodyum klorür ile düzenli yağmurlama ile iç organların kuruma-out önleyin. </li><li> Steril cımbız veya bez kullanarak, dalak ve pankreas dalak lob (kuyruk bölge) açığa superior mide geri çekin. </li><li> Yavaşça visseral periton ile örtülü pankreas baş, boyun ve gövde bölgesini ortaya çıkarmak için sağ üst kadranda üst jejunumun duodenum ve bir kısmını geri çekin. </li></ol></li><li> Ligasyonu <ol><li> Pankreasın boyun bölgesinde pankreas ana kanalının anatomik pozisyonunu bulun. </li><li> Visseral periton ve gastrokolik ligament, retroperitoneal erişim izni pankreas gövde ve kuyruk bölgesini açığa İnsizyon. Boyun bölgesini açığa çıkarmak amacıyla, bir Kocher manevrası gerçekleştirin. Ve duodenum ve baş kaldırınretroperitoneal kapalı pankreas inferior vena kava ve aşağıdaki aort onları yükseltme. </li><li> Dikkatle boyun bölgesine altına spatula yerleştirin. Gastro-duodenal ve dalak lobu ayıran portal ven sol tarafında 6-0 Prolen iplik ile pankreatik kanal Arter. </li><li> Loblar yeterince ayrılmasını temin ilk ligasyon yakın ikinci bir ligasyonu gerçekleştirin. </li><li> Altta yatan kan damarları, yani üstün pancreaticoduodenal arter, alt pancreaticoduodenal arter ve splenik arter pankreas kısmını zarar görmemesi için çok dikkatli Arter. </li><li> Karın boşluğuna geri organları yerleştirin. </li><li> Kas / periton katman için ve cilt için kesintili sütür desen sürekli dikiş desen 4-0 poliglikol ipliksi parçacığı kullanarak kesi kapatın. </li></ol></li></ol> 5. Sham Cerrahisi <ol><li> De tüm adımları uygulayın4.1.4 adımda 1 kayıtsız kalamayız. Pankreas maruz iken, pankreatik kanal ligasyonu yapmazlar. </li><li> Aşama 4.1.4 sonunda geri karın boşluğuna iç organları yerleştirin. </li><li> 4.2.7 açıklandığı gibi kesi kapatın. </li></ol> 6. Post-operasyonel Bakım ve İzleme <ol><li> Cerrahi işlem tamamlandıktan sonra, kurtarma alanında fareyi yerleştirin. Bu, normal vücut sıcaklığının muhafaza edilmesi amacıyla bir ısıtma pedi üzerine yerleştirilir ve düz kağıt yatak ile kaplı bir kafes içerir. </li><li> Kafes altına yerleştirilen nemlendirilmiş gıda post-operatif hipoglisemi önlemek için beslenme desteği sağlamak. Yumuşayana kadar bu amaçla, su ile ıslatılmış standart kemirgen diyeti granül kullanılır. </li><li> Nemlendirilmiş gıda sıvı desteği sağla ve su ad libitum sağlar. </li><li> Ameliyat sonrası 2 gün boyunca günde iki kez - (0.1 mg / kg 0.05) analjezi gibi Buprenophine kullanın. Tüm deneysel sırasında, Gözlem takibie yarasından veya davranış ve aktivite düzeyi damat azalma ile karakterize fiziksel bozulma, düşük iştah ve vücut ağırlığı kaybı sıvı veya irin salgılanması da dahil enfeksiyon olası belirtileri, oluşumu için fareler. </li></ol> Başarılı PDL ve PDL Tail Hasat ve Sham Tail Pankreas 7. Değerlendirme <ol><li> Servikal dislokasyon ile fareler Euthanize. </li><li> Kürk taşınmasını önlemek için karın bölgesini yeniden tıraş. </li><li> Karın deri ve kas tabakası açın ve pankreas iyi erişim elde etmek için deri ve kas geniş bir alanı kaldırmak. </li><li> Steril cımbız veya bez kullanarak, mide, dalak ve pankreas dalak lob (kuyruk bölge) açığa superior çekilir. </li><li> Yavaşça pankreas gastro-duodenal lob (baş bölgesi) ortaya çıkarmak için üst jejunumun duodenum ve bir kısmını dışarı çekin. Not: PDL pankreasın bağlandı bölümünün artık küçültülür ve neredeyse haline gelmiştirsaydam adacıklar küçük beyaz noktalar olarak görünür böylece. Pankreas baş kısmı opak pembe ve görülebilir lobuli farklı exocrine olduğunu. </li><li> Steril makas kullanarak, dalak boyunca keserek, dalak gelen pankreas kuyruk kısmını lige ayırmak. İç organlara pankreas kuyruk bölgesini bağlayan bağ dokusu gevşek kesin. </li><li> Bitişik harfleri ve hasat edilen dokudan buna hemen bitişik doku dışında, hemen ligasyon önünde PDL kuyruk bölgesini kesin. </li><li> Sahte kuyruk doku izole etmek için, adımları 7.5 ile 7.1 izleyin. Kullanımı Steril makas dalak boyunca keserek dalak sahte pankreas kuyruk bölgesini ayrı. İç organlara kuyruk pankreas bağlayan pankreas boyun bölgesi içine kesme ve bağ dokusu gevşek keserek kuyruk bölgesini izole edin Not:, sahte pankreas kuyruk ve baş bölgesi Hem opak pembe ayrı iki parçayı ayırmak için,boyun bölgesinde pankreas kesti. </li></ol>

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The authors have nothing to disclose.

In the present study, we describe in detail the methodology behind PDL, a mouse injury model to study beta cell neogenesis and proliferation and transdifferentiation of pancreatic non-beta cells. Ambiguity in data on PDL among labs stimulates the need for a standardized protocol for PDL surgery.
Critical steps in the PDL protocol include the selection of healthy, young mice for surgery. Preferably male mice should be used since unpublished data from our lab suggest that estrogen receptor signa...

300 milyondan fazla insanlara dünya çapında 1,2 etkileyen diyabet prevalansı artan, yetersiz beta hücre kitlesi doldurmak için, in vitro ve in vivo olarak, hem insülin üreten beta hücrelerinin yeni kaynaklar arama artırmıştır. 3 anahtar belirleme beta hücre çoğalması ve beta hücre neojenezi, örneğin, non-beta hücre veya projenitör hücresi beta hücrelerinin farklılaşmasını düzenleyen mekanizma ve faktörleri, diyabetlerde rejeneratif tedavilerin geliştirilmesi için yeni hedefler sağlayabilir. Gelişmekte olan kemirgen pankreas olarak, endokrin hücre tipleri transkripsiyon faktörü Neurogenin3 (Ngn3) ifade eden, endokrin progenitör hücrelerin geçici popülasyonundan ayırt. 4,5 yetişkin kemirgen pankreas olarak, normal fizyolojik koşullar altında, beta hücre kütlesi optimal sayıda tutulan metabolik ihtiyaçlarını karşılamak için. Beta hücre boyutundaki değişiklikler,beta hücrelerinin potansiyel normal fizyolojik koşullar altında prolifere iken apoptoz ve çoğaltma nüfusun genelinde homojen beta hücre genişlemesi ve dönüş bitti. 6-8 başlıca mekanizmalar oluşturan, 9 onların çoğalması oranı düşüktür ve yeniden çoğaltma sınırlandırıldı yaş ve glukoz metabolizması etkilenir dinamik bir sessizlik dönemi veya refrakter dönem 6,7. 10 endokrin progenitör hücreler kadar normal yetişkin pankreas tespit edilmemiştir beri, neojenezini normal erişkin beta hücre büyümesine katkıda bulunduğu düşünülmektedir. 8 Bu nedenle, genişletilebilir ve bir roman, beta hücrelerinin muhtemelen sınırsız bir kaynak sağlayacak yeni beta hücrelerini verimli yeteneğine sahip yetişkin pankreasta bir fakültatif endokrin progenitör hücre tanımlaması. Kısmi kanalı ligasyonu (PDL) beta hücre neogenesi indüklediği tarif edilmiş bir hayvan yaralanması modeliYetişkin pankreasın s. Bu modelde 11,12, pankreas kuyruk boşaltma ana pankreatik kanal cerrahi bağlanır. ekzokrin drenaj elde edilen tıkanma inflamatuvar ortamında enflamasyon ve ligasyon distal asinar atrofi. 12-14 ile birlikte ana dokuların yeniden indükler, Ngn3 indüklenir endokrin progenitör işaret ve beta hücre hacmi yeniden ekspresyonu, iki kat artar . Embriyonik tip endokrin progenitör hücre ve &quot;sessizlik dönemi&quot; olmadan-çoğaltılması yeniden eğilimli önceden varolan ve yeni kurulan beta hücrelerinin çoğalmasından ifade eden Ngn3 yeni beta hücrelerinin kuşağa beta hücre hacmi sonuçlarında bu iki katına. 11,15 Bu tür pankreatektomi 6,7,16-19 ve beta hücrelerinin 20 seçici ablasyonu gibi yaralanma modellerinde, beta hücre neojenezini ve çoğaltma yoğun tarif edilmiştir. Thes içinde Ancak, rejeneratif sonuçE modelleri hasarın ölçüde etkilenir ve azalmış ilk beta hücre kütlesi 21 ile ilişkilidir. PDL başlangıç ​​beta hücre kütlesi etkilenmez ve beta hücre neojenezini ve proliferasyon sağlam aktive olan bir cerrahi bir modeldir. Gerçekten de, PDL uygulanan farelerin pankreasında, Ngn3 ifade eden hücreler, kanalın epitelial yakınında tespit edilir. Bu hücreler, THP (FACS) kullanılarak Ngn3-GFP transgenik farelerin lige pankreas izole edilir ve E12.5 Ngn3 pankreas ve ex vivo kültür içinde bir nakil aşağıdaki işlevsel p hücrelere karşı ayırt etmek mümkün olabilir - / - . farenin 11 Benzer şekilde, Ngn3 CreERT olarak;. Ngn3 genini aktive hücreler sürekli olarak tamoksifen, enjeksiyon, etiket-pozitif Ngn3 hücresinden türetilmiş beta hücreleri PDL sonra tespit sonra etiketlenir ki burada R26 YFP fareler 15 Ayrıca, yeni oluşan beta hücreleri, önceden seyreltilmiş -existHedef spesifik kısa saç tokası RNA anlamlı beta hücre kitlesi ve beta hücre çoğalmasını sonra azalır kullanarak Ngn3 ifadesini azalmış beri beta hücrelerini ing ve tercihen beta hücrelerinin yüksek çoğalma potansiyeli gösteren, içinde küçük adacıklar bulun. 15 Ngn3 PDL sonra beta hücre genişlemesi için önemli olan PDL. 11 Özellikle, PDL sonra Ngn3 hücresinden türetilmiş beta hücrelerinin fraksiyonu ve beta hücre kütlesi kritik Ngn3 indüksiyonu 15 seviyesine bağlıdır. R26 iDTR fareler;. Bu, Ngn3 Ngn3 CreERT difteri toksin verilmesi ile ifade eden hücrelerin seçici şekilde kesip çıkarılması Ngn3 yüksek ifade seviyesi, pankreas gelişimi sırasında multipotent pankreatik progenitörlerinden endokrin bağlılık için kritik bir adım olduğu gözlemiyle uyumludur ek olarak 22 Özellikle küçük adacıkları azalma insülin içeriğine ve düşük beta hücre çoğalması ile sonuçlanır. 15 <pPDL sonra kanal hücrelerinde Ngn3 ifade indüksiyon birçok 11,15,16,23,24 tarafından onaylandıktan rağmen class = &quot;jove_content&quot;&gt;, PDL sonucuna ilişkin adacıklar hücreleri 24,25 ve tutarsızlıklar içinde Ngn3 ifadesi bizim ilk gözlemler meydan artan beta hücre kitlesi 26,27 arasında, Ngn3 görünümü PDL sonra kanal kaynaklı endokrin atalarıdır 24,26,28,29 ve artan beta hücre çoğalmasını 27 dile getirdi. 30 Bunlar çelişkili sonuçlar, en azından kısmen, en önemlisi fareler, post-operatif fizyolojik ve çevresel koşullar ve analizi, vücut ağırlığı, cinsiyet ve yaş ameliyat sonrası zaman noktalarında varyasyonlar dahil faktörlerin bir kombinasyonu isnat edilebilir Cerrahi teknik farklılıklar. Bizim ellerde 30 beta hücre çoğalması, insülin içeriği, beta hücre hacmi ve küçük adacıklar sayısı sürekli PDL sonra artmıştır. AyrıcaNgn3 mRNA sürekli artar ama biz doğrudan açıklama var olan PDL kuyruk pankreaslarından arasında Ngn3 mRNA ekspresyonu büyük farklılıklar vardır. Biz Ngn3 mRNA seviyesi, beta hücrelerinden 15 gelen beta hücre neojenezi derecesi ile ilişkili olabilir hipotezi, ama, bu da ispat gerektirir. Tüm deneysel varyasyonları kaldırmak olmamasına rağmen, PDL ameliyatı gerçekleştirmek için standart bir yöntem sonuçlarında daha iyi tekdüzelik sağlar ve beta hücre çoğalmasını ve neojenezini okuyan yeni yollar açar.

<table><tbody><tr><td>Supplies for preparation area&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Hibiscrub&amp;nbsp;</td><td>Regent Medical</td><td>5601IE5F11</td><td>Chlorhexidin diglucon</td></tr><tr><td>Ketamin</td><td>Ceva</td><td>BE-V202526</td><td>anesthesia</td></tr><tr><td>Rompun(2%) (Xylazin)</td><td>Bayer</td><td>BE-V041815</td><td>anesthesia</td></tr><tr><td>Duratears</td><td>Alcon</td><td>34335-8</td><td>ointment for eyes</td></tr><tr><td>Razor</td><td></td><td></td><td></td></tr><tr><td>Supplies for surgical area&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Leica Operating microscope</td><td>Leica</td><td>10446320</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools (FST)</td><td>18000-45</td><td></td></tr><tr><td>autoclaved surgical instruments</td><td>Fine Science Tools (FST)</td><td></td><td></td></tr><tr><td>Recirclulating water heating pad</td><td>Gaymar Industries, Inc.</td><td>TP702</td><td></td></tr><tr><td>Adhesive OP-towel</td><td>BARRIER</td><td>706500-07</td><td></td></tr><tr><td>OP-tape</td><td>BARRIER</td><td>381035-00</td><td></td></tr><tr><td>Stella 3/5 Compresse de gaze (Sterile)</td><td>Lohmann&amp;amp;Rauscher International</td><td>35968</td><td></td></tr><tr><td>Mini Plasco 0.9% NaCl solution</td><td>B.BRAUN</td><td>3521680</td><td></td></tr><tr><td>6-0 prolene suture</td><td>Ethicon</td><td>8706H</td><td></td></tr><tr><td>4-0 polyglycol suture</td><td>Ethicon</td><td>SL-607</td><td></td></tr><tr><td>Supplies for recovery area</td><td></td><td></td><td></td></tr><tr><td>Paper bedding (paper towel)</td><td></td><td></td><td></td></tr><tr><td>Vetergesic</td><td>ecuPhar</td><td>BE-V342955</td><td></td></tr><tr><td>Materials for analysis</td><td></td><td></td><td></td></tr><tr><td>Taqman &lt;i&gt;Ngn3&lt;/i&gt; primer</td><td>Integrated DNA technologies</td><td>Mm.PT.56a.33574796.gs</td><td></td></tr><tr><td>Taqman CycloA primer</td><td>Integrated DNA technologies</td><td>Mm.PT.39a.2.gs</td><td></td></tr><tr><td>anti-Rabbit Cleaved Caspase 3 antibody (D175)</td><td>Cell Signaling</td><td>5A1E</td><td></td></tr><tr><td>anti-mouse/rat Ki-67 antibody</td><td>eBioscience</td><td>14-5698-82</td><td></td></tr><tr><td>monoclonal anti-actin alpha-Smooth muscle-Cy3 (mouse)</td><td>Sigma</td><td>085K4889</td><td></td></tr><tr><td>anti-rat Cytokeratin 19&amp;nbsp;</td><td>DSHB</td><td></td><td></td></tr><tr><td>monoclonal Mouse anti-BrdU</td><td>Dako</td><td>M0744</td><td></td></tr><tr><td>Hoechst</td><td>Sigma</td><td>33342</td><td></td></tr></tbody></table>

surgical injury to the mouse pancreas through ligation of the pancreatic duct as a model for endocrine and exocrine reprogramming and proliferation

Bir embriyo veya yetişkin kök hücre farklılaşması ile in vivo veya ex vivo veya beta hücrelerinin üretimi pankreatik beta hücrelerinin genişlemesi, diyabet için bir tedavi olarak, insan doku adacığı transplantasyonundan verici kıtlığı hafifletmek için beta hücrelerinin yeni bir genleşebilen kaynaklar sağlar. Son gelişmeler bu amaca yönelik yapılmış olmasına rağmen, bir kök / progenitör hücreden beta hücre genişlemesi ve farklılaşmasını düzenleyen mekanizmalar karakterize edilmesi gerekmektedir. Burada, doku tekrar modellemesinde ve beta hücre proliferasyonu ve farklılaşmasının mekanizmaları incelemek için bir araç olarak işlev görebilir yetişkin fare pankreası bir yaralanma modeli için bir protokol açıklar. Kısmi kanalı ligasyonu (PDL) pankreas kuyruk bölgesi dışına ekzokrin ürünlerinin drenaj tıkanma sonucu ana pankreatik kanal cerrahi ligasyon içeren kemirgen pankreasın bir deneysel yaralanma olduğunu. tahribatını asiner atrofi, bağışıklık hücre infilt uyarmaktadırrasyon ve şiddetli doku yeniden. Biz daha önce endojen progenitör benzeri hücreler ve PDL sonra beta hücre çoğalmasında bir artış ifade Neurogenin (NGN) 3 aktivasyonunu bildirmiştir. Bu nedenle, PDL beta hücre dinamikleri katılan sinyalleri ve yetişkin pankreas endokrin progenitör özelliklerini incelemek için bir temel sağlar. , Hala faktörler ve yollar PDL beta hücre neojenezi ve çoğalmasına katkıda bulunan, büyük ölçüde belirsizdir yana, PDL için standart protokol laboratuvarlar arasında karşılaştırma için izin verecektir.

PDL asiner atrofi ve inflamasyon indükler ama vücut ağırlığı ve glisemiyi etkilemez Mide ve duodenum bitişiğinde bulunan organın başkanı etkilenmemiş kalırken 8 haftalık erkek BALB / c farelerde, pankreas kuyruk ekzokrin enzimleri drenaj kanalı bağlanır. Yaş, cinsiyet ve kilo eşleştirilmiş erkek BALB / c fareler pankreatik kanal ligasyonu dışında kısmi kanal ligasyonu cerrahisi tüm adımları yansıtan sahte cerrahi, geçmesi. Pankreas dokusu 14, 3...

Watch this Scientific Journal Video about Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation at JoVE.co

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Endokrin ve Ekzokrin yeniden programlanması ve Silahların Yayılmasını Önleme Modeli Olarak Pankreas kanalının bağlanmasını yoluyla Fare Pankreas Cerrahi Sakatlık

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

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Research

JoVE Journal

Medicine

16.1K Görüntüleme.  Vrije Universiteit Brussel. This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation. 

Video: Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Diyabetik Fare Böbrek Kapsül içine, Pankreas Islets Nakli

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Biyoloji

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

Taklit ve 3 boyutlu hücre kültüründe pankreas Acinar-için-duktal metaplazi manipüle

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

Kanser Araştırmaları

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Murine Pankreatik Duktal Adenokarsinomun Ultrason Eşliğinde Ortotopik İmplantasyonu

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

Pankreas Kanserinin Ortotopik Rezeksiyonlu Fare Modeli

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pankreas Kanserini Modellemek için Sinjenik Fare Ortotopik Allogreftleri

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Diyabetik Farenin Epididimal Yağ Tabağında Iskelelerin İskele Destekli Nakli

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Biyomühendislik

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Fare modeli değiştirilmiş manşet tekniği kullanarak pankreas nakli için

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

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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 ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

Biliopankreatik Kanala Sodyum Taurocholate Retrograd Enjeksiyonu Kullanılarak Fare Şiddetli Akut Pankreatit Modelinin Kurulması

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture

Source: Fleming Martinez, A. K. et. al. <a href="https://jove.unicartagenaproxy.elogim.com/v/59096/" target="_blank">Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture</a>. J. Vis. Exp. (2019).
This video describes the technique of differentiating pancreatic acinar cells to ductal cells in ex vivo 3D culture. This helps to understand the mechanisms regulating the acinar-to-ductal metaplasia (ADM), a key process in the early development of pancreatic cancer.

Acinar-to-duktal Metaplazi İndüksiyonu: Acinar Hücreden Duktal Hücreye Farklılaşmayı 3D Ex Vivo Kültürde İncelemek İçin Bir Yöntem

Source: Fleming Martinez, A. K. et. al. Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture. J. Vis. Exp. ...