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

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

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 new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

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 ....

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

1. Provide a dedicated preparation area, a surgical area and a recovery area.
2. Conduct the entire surgical procedure in a laminar flow cabinet to minimize environmental contaminants.
3. Assemble the supplies (as listed in Materials and Methods) needed on the preparation, surgical and recovery area, using proper aseptic technique.
4. Ensure that surgical tools are autoclaved prior to surge....

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 was ha.......

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

List of materials used in this article

Name	Company	Catalog Number	Comments
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 Ki-67 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