Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

Summary

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

Abstract

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Introduction

The pancreas is a bifunctional organ, with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer¹. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology.

A targeted therapeutic delivery method would be beneficial. We have developed a pancreatic duct infusion technique that is an effective and selective method for drug and viral delivery to pancreatic cells. In this model, the pancreatic duct is selectively cannulated, and the common bi....

Protocol

All animal experiments were approved by the Animal Research and Care Committee at the Children's Hospital of Pittsburgh and the University of Pittsburgh Institutional Animal Care and Use Committee.

1. Preoperative Preparation

1. Administer inhaled isoflurane (1 - 3% for maintenance and up to 5% for induction) via a nose cone to an appropriate anesthetic level. Monitor the adequacy of anesthesia by toe pinch with forceps. The lack of a response is considered adequate, and be sure not to overmedicate, as respiratory depression may occur.
2. Place the animal on its back on the dissecting microscope stage, with the abdomen c....

Results

With practice and careful surgical technique, the survival rate of these mice should be greater than 95%. One week after infusion, the desired effect should be evident in the pancreas. Mice at 10 to 12 weeks of age were utilized for pancreatic duct infusions. Here we use an adeno-associated virus serotype 8 (AAV8) with a CMV promoter to express green fluorescent protein (GFP), as compared to a sham surgery. Mice pancreases were harvested 7 days after the infusions. Sections of pancreas, s.......

Discussion

This work describes in detail the methodology behind pancreatic duct infusion, an effective mouse model for the delivery of genes and other molecules specifically and effectively to the pancreas. Ambiguity in the specifics of the procedure between various groups highlighted the need for a standardized protocol^8,9,10.

There are several critical steps of the procedure, beginning with the selection of yo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mice (25 - 30g, 8-12 weeks old)	Jackson Laboratory
Ketofen	Henry Schein Inc.	5487
Ethanol (70%)	Sigma-Aldrich	34923
Sterile Saline Solution	Baxter Healthcare Corp.	2B-13-00
Protective Equipment
Hair Removal Product			Nair or trimmer
Gauze Pads 2in x 2in	Fisher Healthcare	22-362-178
Needles (30.5G and 25G)	Becton Dicksinson and Co.	305106 and 305122
Syringe for Ketofen and Saline	Becton Dicksinson and Co.	309657
Syringe for Pump	Henke Sass Wolf	4010.200V0
Infusion Pump	Pump Systems Inc.	NE-1000
Infusion Catheter	World Precision Instruments	CMF31G
Curved Bulldog Clamps (2)	Roboz	RS-7439
Arruga Forceps	Roboz	RS-5163
Adson Forcep	Roboz	RS-5234
Needle Holder	Roboz	RS-7882
Scissor	Roboz	RS-5982
Cotton Tip Applicators	Physician Sales and Services	22-9988
Dissecting Microscope
Suture	Owens and Minor	SXMD1B402