Хирургическое Травма поджелудочной железы мыши через перевязка панкреатического протока в качестве модели для эндокринной и экзокринной перепрограммирования и распространения

Summary

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.

Abstract

Расширение панкреатических бета-клетках в естественных условиях или в силу естественных или поколения бета-клеток путем дифференцирования из эмбриона или взрослого стволовых клеток, может обеспечить новые расширяемые источники бета-клеток, чтобы облегчить дефицит доноров в трансплантации островков человека в качестве терапии для лечения диабета. Хотя недавние успехи были достигнуты к достижению этой цели, механизмы, регулирующие расширение бета и дифференцировку клеток из стволовых клеток / клеток-предшественников еще предстоит характеризуется. Здесь мы опишем протокол для модели повреждения в поджелудочной железе взрослых мышей, которые могут функционировать в качестве инструмента для изучения механизмов ремоделировании ткани и пролиферации бета-клеток и дифференциации. Частичное канал перевязки (PDL), является экспериментально индуцированной травмы поджелудочной железы грызунов участием хирургическое лигирование главного панкреатического протока в результате обструкции дренажа экзокринных продуктов из области хвоста поджелудочной железы. Нанесли ущерб вызывает атрофию ацинарных, иммунную infilt клетокРацион и тяжелой ткани ремоделирования. Ранее мы уже сообщали активации Neurogenin (NGN) 3, выражая эндогенных предшественников, как клетки и увеличение пролиферации бета-клеток после PDL. Таким образом, PDL обеспечивает основу для изучения сигналов, участвующих в динамике бета-клеток и свойства эндокринной прародителя во взрослой поджелудочной железы. Так, он по-прежнему остается в значительной степени неясным, какие факторы и пути способствуют регенерации бета-клеток и пролиферации в PDL, стандартизированный протокол для PDL позволит для сравнения между лабораториями.

Introduction

The increasing prevalence of diabetes, affecting more than 300 million people world-wide^1,2 has boosted the search for new sources of insulin-producing beta cells, both in vitro and in vivo, to replenish the deficient beta cell mass.³ Identifying key mechanisms and factors that regulate beta cell proliferation and beta cell neogenesis, i.e., the differentiation of beta cells from a non-beta cell or progenitor cell, can provide novel targets for the development of regenerative therapies in diabetes.

In the developing rodent pancreas, all of the endocrine cell types differentiate from a transient population of endocrine progenitor cells, expressing the transcription factor Neurogenin3 (Ngn3).^4,5 In the adult rodent pancreas, under normal physiological conditions, the beta cell mass is maintained at an optimal number to meet metabolic demands. Changes in beta cell size, apoptosis and replication constitute the major mechanisms for beta cell expansion and turn-over.^6-8 While the potential of beta cells to proliferate under normal physiological conditions is homogenous throughout the population,⁹ their proliferation rate is low and re-replication is restricted by a dynamic quiescence period or refractory period^6,7, influenced by age and glucose metabolism.¹⁰ Since endocrine progenitor cells have so far not been identified in the normal adult pancreas, neogenesis is thought to not contribute to normal adult beta cell growth.⁸

Therefore, the identification of a facultative endocrine progenitor cell in the adult pancreas that is expandable and capable of yielding new beta cells would provide a novel, possibly unlimited source of beta cells.

Partial duct ligation (PDL) is an animal injury model that has been described to induce beta cell neogenesis in the adult pancreas.^11,12 In this model, the main pancreatic duct draining the pancreatic tail is surgically ligated. The resulting obstruction of exocrine drainage induces major tissue remodeling, accompanied by inflammation and acinar atrophy distal to the ligation.^12-14 Within this inflammatory environment, re-expression of the endocrine progenitor marker Ngn3 is induced and the beta cell volume increases two-fold. This doubling in beta cell volume results from the generation of new beta cells from an Ngn3 expressing embryonic-type endocrine progenitor cell and from proliferation of pre-existing and newly-formed beta cells that are prone to re-duplication without “quiescence period”.^11,15

Beta cell neogenesis and replication in injury models, such as pancreatectomy^6,7,16-19 and selective ablation of beta cells²⁰ have been extensively described. However, the regenerative outcome in these models is influenced by the extent of the inflicted damage and is associated with a decreased initial beta cell mass²¹. PDL is a surgical model in which the initial beta cell mass is not affected and beta cell neogenesis and proliferation are robustly activated. Indeed, in the pancreas of mice that underwent PDL, Ngn3 expressing cells are identified near the epithelial lining of the duct. These cells can be isolated from the ligated pancreas of Ngn3-GFP transgenic mice using fluorescence activated cell sorting (FACS) and are able to differentiate towards functional beta cells following engraftment into and ex vivo culture of the pancreas of E12.5 Ngn3^-/- mice.¹¹ Similarly, in Ngn3^CreERT;R26^YFP mice in which cells that activated the Ngn3 gene are permanently labeled after tamoxifen injection, label-positive Ngn3 cell-derived beta cells are detected after PDL.¹⁵ Moreover, newly formed beta cells dilute pre-existing beta cells and preferentially locate in small islets within which beta cells show high proliferation potential.¹⁵ Ngn3 is important for beta cell expansion after PDL since decreased Ngn3 expression using target-specific short-hairpin RNA significantly decreases beta cell mass and beta cell proliferation after PDL.¹¹ Notably, the fraction of Ngn3 cell-derived beta cells and the beta cell mass after PDL critically depends on the level of Ngn3 induction¹⁵. This is in accordance with the observation that high level of Ngn3 expression is a critical step for endocrine commitment from multipotent pancreatic progenitors during pancreatic development.²² In addition, selective ablation of Ngn3 expressing cells by diphtheria-toxin administration to Ngn3^CreERT;R26^iDTR mice results in decreased insulin content and reduced beta cell proliferation, especially in small islets.¹⁵

Although the induction of Ngn3 expression in duct cells after PDL has been confirmed by many^{11,15,16,23,24}, Ngn3 expression in islets cells^24,25 and discrepancies in outcome of PDL challenged our initial observations of increased beta cell mass^26,27, appearance of Ngn3 expressing duct-derived endocrine progenitors^24,26,28,29 , and increased beta cell proliferation²⁷ after PDL.³⁰

These conflicting results could, at least partially, be attributed to a combination of factors, including variations in the post-surgical time points of analysis, bodyweight, sex and age of the mice, post-operative physiological and environmental conditions and, most importantly, differences in surgical technique.³⁰ In our hands, beta cell proliferation, insulin content, beta cell volume and the number of small islets are consistently increased after PDL. Also Ngn3 mRNA consistently increases, but there are large differences in the Ngn3 mRNA expression between PDL tail pancreases, for which we have no direct explanation. We hypothesized that the level of Ngn3 mRNA might correlate with the degree of beta cell neogenesis from non-beta cells¹⁵, but this needs further substantiation. Although it does not remove all experimental variations, a standardized method for performing PDL surgery allows for better uniformity in results and opens new avenues in studying beta cell proliferation and neogenesis.

Protocol

All manipulations follow the guidelines issued by the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123 and and 2010/63/EU). 1. Preparation of Work Area Provide a dedicated preparation area, a surgical area and a recovery area. Conduct the entire surgical procedure in a laminar flow cabinet to minimize environmental contaminants. Assemble the supplies (as listed in Materials and Methods) nee…

Representative Results

PDL induces acinar atrophy and inflammation but does not affect bodyweight and glycemia In 8 week old male BALB/c mice, the duct draining the exocrine enzymes from the tail of the pancreas is ligated while the organ’s head, located adjacent to the stomach and duodenum, remains unaffected. Age, sex and weight-matched male BALB/c mice undergo sham surgery recapitulating all steps of partial duct ligation surgery, except the ligation of the pancreatic duct. Pancreas tissue …

Discussion

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…

Materials

Supplies for preparation area
Hibiscrub	Regent Medical	5601IE5F11	Chlorhexidin diglucon
Ketamin	Ceva	BE-V202526	anesthesia
Rompun(2%) (Xylazin)	Bayer	BE-V041815	anesthesia
Duratears	Alcon	34335-8	ointment for eyes
Razor
Supplies for surgical area
Leica Operating microscope	Leica	10446320
Hot bead sterilizer	Fine Science Tools (FST)	18000-45
autoclaved surgical instruments	Fine Science Tools (FST)
Recirclulating water heating pad	Gaymar Industries, Inc.	TP702
Adhesive OP-towel	BARRIER	706500-07
OP-tape	BARRIER	381035-00
Stella 3/5 Compresse de gaze (Sterile)	Lohmann&Rauscher International	35968
Mini Plasco 0,9% NaCl solution	B.BRAUN	3521680
6-0 prolene suture	Ethicon	8706H
4-0 polyglycol suture	Ethicon	SL-607
Supplies for recovery area
Paper bedding (paper towel)
Vetergesic	ecuPhar	BE-V342955
Materials for analysis
Taqman Ngn3 primer	Integrated DNA technologies	Mm.PT.56a.33574796.gs
Taqman CycloA primer	Integrated DNA technologies	Mm.PT.39a.2.gs
anti-Rabbit Cleaved Caspase 3 antibody (D175)	Cell Signaling	5A1E
anti-mouse/rat Ki67 antibody	eBioscience	14-5698-82
monoclonal anti-actin alpha-Smooth muscle-Cy3 (mouse)	Sigma	085K4889
anti-rat Cytokeratin 19	DSHB
monoclonal Mouse anti-BrdU	Dako	M0744
Hoechst	Sigma	33342