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).

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
<ol>
	<li>Provide a dedicated preparation area, a surgical area and a recovery area.</li>
	<li>Conduct the entire surgical procedure in a laminar flow cabinet to minimize environmental contaminants.</li>
	<li>Assemble the supplies (as listed in Materials and Methods) needed on the preparation, surgical and recovery area, using proper aseptic technique.</li>
	<li>Ensure that surgical tools are autoclaved prior to surgery.</li>
	<li>Provide a recirculating water heating pad at a temperature of 38 &#176;C for temperature stabilization during surgery. Cover the heating pad with a sterile waterproof pad.</li>
	<li>Provide an operating microscope with a magnification of at least 6.3X.</li>
	<li>Use an instrument sterilizer, such as a hot bead sterilizer, to sterilize instruments in between surgical procedures.</li>
	<li>Prepare a recovery area consisting of a large cage, lined by flat paper bedding</li>
</ol>

2. Preparing the Animals for Surgery
<ol>
	<li>For PDL and sham surgery, use 8 week old male mice, housed in standard cages and maintained on a 12 hr&#160;light/12 hr&#160;dark cycle and fed a standard rodent diet ad libitum. 
	Note: Here, we use BALB/cJrJ mice but we have also successfully used other strains and various transgenic strains.</li>
	<li>Use Buprenophine as preemptive analgesia (0.05 &#8211; 0.1 mg/kg) 30 min prior to surgery.</li>
	<li>Anesthetize the mice by intraperitoneal injection of 100 mg/kg of ketamine and 5 - 10 mg/kg of xylazine.</li>
	<li>Assess proper anesthetization by observing gradual loss of voluntary movement and muscle relaxation. Test the loss of reflexes by toe pinching.</li>
	<li>Apply ophthalmic ointment to prevent dryness of the eyes while under anesthesia.</li>
</ol>
3. Surgical Site Preparation
<ol>
	<li>Disinfect thorax and abdomen with antiseptic chlorhexidine solution.</li>
	<li>Shave an area of 2.5 cm x 1.5 cm of the abdomen.</li>
	<li>Disinfect the shaven area using gauze soaked with chlorhexidine solution, then alcohol solution and a final application of chlorhexidine solution.</li>
	<li>Position the animal in the surgical area so that the prepared surgical site is upwards facing the surgeon.</li>
	<li>Drape the mouse using a waterproof surgical drape with an opening that leaves the disinfected abdominal region exposed while covering the rest of the body to create a sterile working field. Monitor the mouse prior to the procedure for depth of anesthesia.</li>
</ol>
4. Pancreatic Duct Ligation
<ol>
	<li>Laparotomy
	<ol>
		<li>Make an upper midline incision in the skin extending from the xiphoid process to the umbilicus using a sterile surgical blade. Separate the underlying linea alba and the peritoneum using sterile scissors in order to expose the upper abdominal quadrant.</li>
		<li>Prevent drying-out of the internal organs by regular sprinkling with sterile 0.9% sodium chloride.</li>
		<li>Using sterile tweezers or swab, retract the stomach superiorly, exposing the spleen and the splenic lobe (the tail region) of the pancreas.</li>
		<li>Gently retract the duodenum and part of the upper jejunum to the right upper abdominal quadrant to expose the head, neck and body region of the pancreas covered by the visceral peritoneum.</li>
	</ol>
	</li>
	<li>Ligation
	<ol>
		<li>Locate the anatomical position of the pancreatic main duct in the neck region of the pancreas.</li>
		<li>Incise the visceral peritoneum and the gastrocolic ligament, granting access to the retroperitoneum, exposing the body and tail region of the pancreas. In order to expose the neck region, perform a Kocher maneuver. Lift the duodenum and head of the pancreas off the retroperitoneum elevating them from the inferior vena cava and aorta below.</li>
		<li>Carefully place the spatula underneath the neck region. Ligate the pancreatic duct with 6-0 Prolene thread at the left side of the portal vein that separates the gastro-duodenal and splenic lobes.</li>
		<li>Perform a second ligation in close proximity to the first ligation to ensure that the lobes are adequately separated.</li>
		<li>Ligate very carefully in order not to damage the underlying blood vessels, namely the superior pancreaticoduodenal artery, the inferior pancreaticoduodenal artery and the pancreatic part of the splenic artery.</li>
		<li>Place the organs back into the abdominal cavity.</li>
		<li>Close the incision using 4-0 polyglycol filamentous thread in a continuous suture pattern for the muscle/peritoneal layer and in a discontinuous suture pattern for the skin.</li>
	</ol>
	</li>
</ol>
5. Sham Surgery
<ol>
	<li>Perform all steps as described in steps 1 through 4.1.4. While the pancreas is exposed, do not perform ligation of the pancreatic duct.</li>
	<li>At the end of step 4.1.4, place internal organs back into the abdominal cavity.</li>
	<li>Close the incision as described in 4.2.7.</li>
</ol>
6. Post-operational Care and Monitoring
<ol>
	<li>After the surgical procedure is complete, place the mouse in the recovery area. This consists of a cage placed on a heating pad and lined with flat paper bedding in order to maintain normal body temperature.</li>
	<li>Provide nutritional support to avoid post-operative hypoglycemia by moistened food placed on the cage bottom. For this purpose, use standard rodent diet pellets soaked in water until they soften.</li>
	<li>Provide fluid support by the moistened food and provide water ad libitum.</li>
	<li>Use Buprenophine as analgesia (0.05 &#8211; 0.1 mg/kg) twice daily for 2 days post-surgery. During the entire experimental, follow up, observe the mice for occurrence of possible signs of infection, including secretion of liquid or pus from the wound, or for physical deterioration characterized by reduction in grooming behavior and activity level, lower appetite and bodyweight loss.</li>
</ol>
7. Evaluation of Successful PDL and Harvest of PDL Tail and Sham Tail Pancreas
<ol>
	<li>Euthanize mice by cervical dislocation.</li>
	<li>Re-shave the abdominal region to avoid carry-over of fur.</li>
	<li>Open the abdominal skin and muscle layer and remove a large area of skin and muscle to obtain good access to the pancreas.</li>
	<li>Using sterile tweezers or swab, the stomach is retracted superiorly, exposing the spleen and the splenic lobe (the tail region) of the pancreas.</li>
	<li>Gently pull out the duodenum and part of the upper jejunum to expose the gastro-duodenal lobe (head region) of the pancreas. 
	Note: The ligated portion of the PDL pancreas has now reduced in size and has become almost translucent so that islets are visible as small white dots. The head portion of the pancreas is opaque pink and distinct exocrine lobuli can be observed.</li>
	<li>Using sterile scissors, separate the ligated tail part of the pancreas from the spleen, by cutting along the spleen. Cut loose the connective tissue connecting the tail region of the pancreas to the internal organs.</li>
	<li>Cut the PDL tail region right in front of the ligation, excluding the ligature and the tissue immediately adjacent to it from the harvested tissue.</li>
	<li>To isolate sham tail tissue, follow steps 7.1 through 7.5. Using sterile scissors separate the tail region of the sham pancreas from the spleen by cutting along the spleen. Isolate the tail region by cutting into the neck region of the pancreas and cutting loose the connective tissue connecting the tail pancreas to the internal organs 
	Note: Both the tail and head region of the sham pancreas are opaque pink, to isolate both parts separately, cut the pancreas in the neck region.</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...

The increasing prevalence of diabetes, affecting more than 300 million people world-wide1,2 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 period6,7, 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 &#8220;quiescence period&#8221;.11,15
Beta cell neogenesis and replication in injury models, such as pancreatectomy6,7,16-19 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 Ngn3CreERT;R26YFP 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&#160;Ngn3&#160;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 Ngn3CreERT;R26iDTR 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 many11,15,16,23,24, Ngn3 expression in islets cells24,25 and discrepancies in outcome of PDL challenged our initial observations of increased beta cell mass26,27, appearance of Ngn3 expressing duct-derived endocrine progenitors24,26,28,29 , 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.

<table border="0" cellpadding="0" cellspacing="0">
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		<tr>
			<td>Supplies for preparation area&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hibiscrub&#160;</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies for surgical area&#160;</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&#38;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></td>
			<td></td>
			<td></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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman Ngn3 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 Ki67 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&#160;</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

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.

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&#8217;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 ...

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.

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 ...

surgical-injury-to-mouse-pancreas-through-ligation-pancreatic-duct-as

Research

JoVE Journal

Medicine

16.1K visualizações.  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. 

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

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

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.

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

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.

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, ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The recent rise of robotic and laparoscopic pancreatic surgery has found benefits such as reduced time to functional recovery. Few studies have reported on the feasibility, technique and outcome of robotic LPJ, especially including transection of the gastroduodenal artery. The present study describes the main steps for robotic LPJ in a patient with painful chronic pancreatitis with a dilated main pancreatic duct. The patient underwent robotic LPJ. The LPJ anastomosis is performed using a running suture technique in a longitudinal side-to-side manner. Routinely, the gastroduodenal artery is transected to drain the entire length of the main pancreatic duct. The patient is in French position; 7 trocars are placed (4 robotic, 2 laparoscopic assistants, 1 liver-retractor). After docking of the robot system, the omental bursa is opened, and the right gastroepiploic artery and vein are ligated at their base at the lower border of the pancreas. Intraoperative ultrasonography is performed in order to determine trajectory of the dilated main pancreatic duct which is opened for its entire length after the gastroduodenal artery has been suture ligated both cranially and caudally from the main pancreatic duct. A Roux limb is created, and a latero-lateral PJ is fashioned using several 3-0 barbed sutures. A stapled jejuno-jejunostomy is created at sufficient distance from the pancreatic anastomosis, aided by a 50 cm suture. The described technique for robotic LPJ is a complex but feasible operation for patients with treatment refractory CP and a dilated main pancreatic duct. Due to its complexity, implementation in high volume centers with extensive experience with CP surgery may improve outcomes.

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal artery.

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

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.

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