The authors have no acknowledgements. Funding was received from the Foundation of Health and Family Planning Commission of Zhejiang Province (Grant 20146242) and the Foundation of Wenzhou Science &amp; Technology Bureau (Grant Y20140248).

All animal experiments were approved by the Animal Research and Care Committee at the Children&#39;s Hospital of Pittsburgh and the University of Pittsburgh Institutional Animal Care and Use Committee.
1. Preoperative Preparation
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	<li>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 n...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+the+pancreatic+lineage:+NGN3++cells+are+islet+progenitors+and+are+distinct+from+duct+progenitors.">Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.</a> Development. 129 (10), 2447-2457 (2002).</li><li>Weinberg, N., Ouziel-Yahalom, L., Knoller, S., Efrat, S., Dor, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+tracing+evidence+for+in+vitro+dedifferentiation+but+rare+proliferation+of+mouse+pancreatic+beta-cells.">Lineage tracing evidence for in vitro dedifferentiation but rare proliferation of mouse pancreatic beta-cells.</a> Diabetes. 56 (5), 1299-1304 (2007).</li><li>Raper, S. E., DeMatteo, R. 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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 protocol8,9,10.
There are several critical steps of the procedure, beginning with the selection of yo...

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 bile duct is occluded, allowing for delivery exclusively to the pancreas.
This technique can allow for the lineage tracing of specific cell types to further elucidate the developmental pathways important to pancreatic development and pathology2,3. Different promoters in viral vectors allow for the specific targeting of cell lines and for the introduction of genes into these cell lines4,5. This work demonstrates 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.

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Mice (25-30g, 8-12 weeks old)</td>
			<td style="width:171px;">Jackson Laboratory</td>
			<td style="width:137px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Ketofen</td>
			<td style="width:171px;">Henry Schein Inc.</td>
			<td style="width:137px;">5487</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Ethanol (70%)</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:137px;">34923</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Sterile Saline Solution</td>
			<td style="width:171px;">Baxter Healthcare Corp.</td>
			<td style="width:137px;">2B-13-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Protective Equipment</td>
			<td style="width:171px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Hair Removal Product</td>
			<td style="width:171px;"></td>
			<td style="width:137px;"></td>
			<td>Nair or trimmer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Gauze Pads 2in x 2in</td>
			<td style="width:171px;">Fisher Healthcare</td>
			<td style="width:137px;">22-362-178</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Needles (30.5G and 25G)</td>
			<td style="width:171px;">Becton Dicksinson and Co.</td>
			<td style="width:137px;">305106 and 305122</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Syringe for Ketofen and Saline</td>
			<td style="width:171px;">Becton Dicksinson and Co.</td>
			<td style="width:137px;">309657</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Syringe for Pump</td>
			<td style="width:171px;">Henke Sass Wolf</td>
			<td style="width:137px;">4010.200V0</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Infusion Pump</td>
			<td style="width:171px;">Pump Systems Inc.</td>
			<td style="width:137px;">NE-1000</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:288px;">Infusion Catheter</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:137px;">CMF31G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Curved Bulldog Clamps (2)</td>
			<td style="width:171px;">Roboz</td>
			<td style="width:137px;">RS-7439</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Arruga Forceps</td>
			<td style="width:171px;">Roboz</td>
			<td style="width:137px;">RS-5163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Adson Forcep</td>
			<td style="width:171px;">Roboz</td>
			<td style="width:137px;">RS-5234</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:288px;">Needle Holder</td>
			<td style="width:171px;">Roboz</td>
			<td>RS-7882</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:288px;">Scissor</td>
			<td style="width:171px;">Roboz</td>
			<td>RS-5982</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Cotton Tip Applicators</td>
			<td style="width:171px;">Physician Sales and Services</td>
			<td>22-9988</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dissecting Microscope</td>
			<td style="width:171px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Suture</td>
			<td style="width:171px;">Owens and Minor</td>
			<td>SXMD1B402</td>
			<td></td>
		</tr>
	</tbody>
</table>

pancreatic duct infusion: an effective and selective method of drug and viral delivery

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.

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...

Watch this Scientific Journal Video about Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery at JoVE.com

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

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.

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

The overall goal of this pancreatic duct infusion procedure is to enable lineage tracing, gene introduction, and cell line-specific targeting in the pancreas. This method could help answer key questions about diseases affecting the pancreas, including chronic pancreatitis, diabetes, and pancreatic cancer. To begin, place an anesthetized mouse under a dissecting microscope so that its abdomen is positioned in the center of the field of view.Immobilize the animal with a surgical tape and then apply an ophthalmic ointment to its eyes to prevent them from drying. Next, cover the abdomen of the mouse with a thick layer of hair removal cream. And after approximately three minutes, gently wipe off the cream with gauze soaked in 70%ethanol to remove all hair.Finally, wipe the skin with 70%ethanol, followed by 10%povidone iodide solution to remove any contaminants and disinfect the skin. To begin the surgery, create with a scalpel a midline skin incision extending from the xiphoid process to the umbilicus. Then using Adson forceps, lift the peritoneum and make with scissors a small midline hole.Expand the cut along the midline of the peritoneum so that its length is equal to the length of the skin incision. Next, use blunt retractors to pull up the liver and after exposing the common bile duct, clamp it with a curved bulldog vascular clamp. Identify the duodenum and with Arruga forceps, pull it out caudally to display the papilla visible as a white spot on the anterior surface of the second portion of the duodenum.To identify the pancreatic duct, withdraw the duodenum caudally, then position a 30-1/5 gauge needle tangentially to the duodenum and make a puncture on its wall that directly opposites the papilla. To infuse the solution, introduce the catheter connected to an infusion pump through the created duodenotomy and slide it along the duct until it is visible within the vessel, but reaches not further than one centimeter to avoid bypassing minor ductal tributaries. Immobilize the catheter by clamping it with a second curved bulldog vascular clamp.Then turn on the pump to initiate the infusion of 150 microliters of the solution into the duct. Next, inject to the peritoneum of the mouse approximately 1.5 milliliters of saline to avoid any loss of infused volume and then cover the exposed bowel with a moistened gauze to prevent desiccation. Once the infusion is completed, release the immobilized catheter and using the bulldog clamp, gently remove it from the pancreatic duct.Then release the clamp from the common bile duct. Close the peritoneum and the skin by continuously suturing them in a single layer and administer the first of the two doses of analgesic, the second of which will be given the following day. Then return the mouse to a cage positioned under a heat lamp and provide the animal with food and water ad libitum.Observe the mouse until it has regained sufficient consciousness manifested in sternal recumbency. Seven days after the surgery, excise from the euthanized animal pancreas as well as spleen, liver, and duodenum serving as controls. Immerse the entire isolated tissues in a 4%paraformaldehyde solution and fix them at four degrees Celsius overnight.Once the tissues are fixed, cryo protect the samples by soaking them in a 30%sucrose solution at four degrees Celsius overnight. After an overnight cryo protection, snap freeze the samples in liquid nitrogen and using a microtome, cut tissue sections at six micrometers. Finally, image the sections under a fluorescent microscope.Presented here are microscopic images of pancreas sections excised from mice seven days after sham surgery and pancreatic duct infusion of AAV8 carrying the GFP gene. Liver, spleen, and duodenum isolated from the mouse infused with AAV8 did not show any GFP signal while the pancreas revealed prominent GFP expression, confirming pancreas-specific delivery of the virus following pancreatic duct infusion. Once mastered, this technique can be done in 10 minutes when performed properly.While performing this procedure, it's very important to perform careful dissection and make the smallest possible duodenotomy for introduction of the catheter. After its development, the technique paved the way for researchers studying the pancreas to investigate a number of pathologies that afflict the organ, including chronic pancreatitis, diabetes, and pancreatic cancer in both rodent and larger animal models. After watching this video, you should have a very good understanding of how to both effectively and selectively infuse the pancreatic duct.

pancreatic-duct-infusion-an-effective-selective-method-drug-viral

Research

JoVE Journal

Medicine

4.4K visualizações.  Children's Hospital of Pittsburgh of UPMC. O objetivo geral deste procedimento de infusão de dutos pancreáticos é permitir o rastreamento de linhagem, introdução genética e direcionamento específico da linha celular no pâncreas. Esse método pode ajudar a responder a perguntas-chave sobre doenças que afetam o pâncreas, incluindo pancreatite crônica, diabetes e câncer de pâncreas. Para começar, coloque um rato anestesiado sob um microscópio dissecando para que seu abdômen esteja posicionado no centro do campo de visão....