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 signaling affects the outcome of PDL. PDL surgery induces recruitment and expression of many factors to/in the injured pancreas and dissection of this cocktail may allow the identification of factors that are necessary and sufficient to cause beta cell neogenesis and proliferation to bypass the need for surgery. However, it is important to realize that the cytokines and factors involved in this model are not yet completely characterized. Therefore, it is important to perform PDL surgery under optimal conditions since severe infection or disease of the animal can alter the occurrence of these factors and thereby affect the outcome of PDL. We advise to monitor the animals closely after PDL surgery by assessing weight, glycemia, appetite, and activity level.
Proper ligation of the pancreatic duct is the most crucial step in the protocol. The ligation needs to be secured with multiple loops to prevent incomplete ligation that may reduce the severity of the injury. However, the ligation should only affect the pancreatic duct, avoiding the underlying superior and inferior pancreaticoduodenal artery. Accidental ligation of the arteries can lead to accessory damage and to hypoxia of the pancreas with possible subsequent necrosis. 
A sub-optimal PDL can occur due to improper ligation of the duct. This leads to a remnant of acinar tissue, as evidenced at day 7 and 14 after PDL surgery. We advise not to include sub-optimal PDL samples into the analysis, since this can lead to variation in analyzed parameters such as beta cell proliferation and Ngn3 expression. Notably, the level of Ngn3 expression correlates with the level of beta cell neogenesis.15 [Hansen M.T., et al., in preparation]. A successful PDL can be recognized by the visible reduction in size and transparency of the tissue compared to a non-ligated pancreas, phenomena which are clearly visible at 7 and 14 days post-surgery. Histological analysis should reveal fibrotic tissue, increased number of ductal structures, increased ductal and endocrine proliferation and recruitment of immune cells. RT-qPCR should reveal an expression of Ngn3 in PDL tail of at least 20% of the expression level in duodenum.
When analyzing proliferation in a non-automated manner by individual inspection of cells, the duct and endocrine cell proliferation in PDL leads to very reproducible results. The induction of Ngn3 expression, however, varies between experiments, indicating that this process may be more sensitive to physiological and environmental factors. Since PDL induces recruitment and/or activation of many cells and factors, identification of the factor(s) responsible for a certain process in PDL is challenging. 
As a model in diabetes research, PDL does not require removal of pancreatic tissue or of beta cell reduction, as compared to pancreatectomy, alloxan or streptozotocin injection. Therefore, animals usually maintain good glycemic control after PDL surgery. It can be expected that beta cell proliferation and activation of an Ngn3-expressing progenitor is stimulated by locally produced signals originating from the inflammatory responses and from acinar cell death. Therefore, PDL is an injury model that can serve as an analytical tool to study signaling pathways involved in beta cell formation. Moreover, the experimental model of PDL may be useful beyond diabetes research: while being artificial, the inflammatory nature of PDL mimics human pathological conditions and has therefore been used as a model to study pancreatitis31, formation of adenocarcinoma32 and neoplasia.33

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><tbody><tr><td>Supplies for preparation area&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Hibiscrub&amp;nbsp;</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>Supplies for surgical area&amp;nbsp;</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&amp;amp;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>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>Materials for analysis</td><td></td><td></td><td></td></tr><tr><td>Taqman &lt;i&gt;Ngn3&lt;/i&gt; 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 Ki-67 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&amp;nbsp;</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 was harvested 3, 7, 14, 30 days post-surgery.
When PDL is performed correctly, mice appear healthy and do not show a significant difference in bodyweight (Figure 1A) or glycemia (Figure 1B) compared to sham-operated mice. Exocrine acinar tissue is lost gradually after PDL surgery (Figure 2A-E), resulting in a reduction in size and weight of the ligated portion of the PDL pancreas (Figure 3), from hereon called PDL tail. Three days post PDL, acinar tissue morphology becomes disrupted and acinar cells undergo apoptosis (Figure 4) and are likely engulfed by infiltrating CD45+ immune cells (Figure 5). At day 7 post PDL, many acinar lobules are replaced by fibrotic (Figure 6) and adipose tissue (Figure 2C-E) while remaining acinar cells undergo acinar-to-ductal metaplasia. By day 14 post PDL, almost all acinar lobuli are devoid of acinar cells (Figure 2A-E) making the PDL tail pancreas appear translucent so that islets become visible to the naked eye (Figure 3). Coincident with the initiation of acinar apoptosis, an increase in cell cycle activity of the ductal epithelium is observed (Figure 7).
PDL induces an increase in insulin+ beta cell volume
Two weeks after PDL the total beta cell volume in PDL tail has doubled as compared to non-ligated Sham tail. Beta cell volume is quantified by measuring the INS+ area in 4 &#181;m sections, 36 &#181;m apart spanning the whole tissue accounting for 10% of the total pancreas volume. PDL induces an increase in insulin content two weeks after surgery compared to non-ligated pancreas as can be shown by automated whole &#8211;tissue optical tomography (OPT).15 Since beta cell size is not changed after PDL the increase in beta cell volume is the result of an increase in beta cell number.
PDL induces an increase in beta cell proliferation and the activation of Ngn3 expression
Beta cell proliferation is analyzed by immunohistochemical (IHC) staining in pancreata harvested 7 days and 14 days after Sham or PDL surgery. The percentage of beta cells positive for proliferation marker Ki-67 is quantified by inspection of individual cells in a non-automated manner. At 7 and 14 days post PDL surgery, beta cell proliferation in PDL tail is significantly increased compared to non-ligated pancreas.
Within 3 days after PDL the expression of the embryonic islet progenitor marker Ngn3 is significantly increased in PDL tail as compared to non-ligated pancreas. Maximal levels of Ngn3 transcript were reached within 1 week and subsequently decreased slowly. We routinely measure Ngn3 gene activation by expressing the level of Ngn3-encoding mRNA in PDL pancreas relative to the stable Ngn3 level in duodenum. We recently suggested that the extent of neogenesis in PDL might depend on the level of Ngn3 gene activation.15,30
<img alt="Figure 1" src="/files/ftp_upload/52765/52765fig1.jpg" /> 
Figure 1. PDL does not affect bodyweight or glycemia. Bodyweight(g) (A) and glycemia (mg/dL) (B) from sham-operated (white bars) and PDL mice (black bars) was measured at different time points (day 0, 7, 14 and 30) following surgery and did not show any significant difference between sham and PDL operated mice at any time point. Figure originally published by Xu et al., 2008.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52765/52765fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52765/52765fig2.jpg" /> 
Figure 2. PDL leads to a gradual loss of acinar cells. Morphological change of PDL tail pancreas revealed by haematoxylin-eosin staining at day 3 (B), 7 (C), 14 (D) and 30 (E) post surgery, compared to sham-operated tail pancreas (A). At 3 days post ligation, only a subtle disruption of the acinar tissue can be observed (B). At 7 days post ligation, acinar tissue is severely disrupted and ductal complexes have formed (C). At 14 days post ligation, few acinar cells remain and acinar lobuli are replaced by ductal structures, a fibrous network and adipocytes (indicated with an asterisk (*) in panel C and E). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52765/52765fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52765/52765fig3.jpg" /> 
Figure 3. PDL tail at harvest.&#160;Sham tail (A) and PDL tail (B) harvested at day 14 post-surgery. PDL tail is dramatically reduced in size compared to Sham tail. Picture (C) shows PDL tail at day 14 post surgery in situ. Due to loss of acinar cells, the PDL tail appears translucent, the main pancreatic duct is clearly visible (indicated with black arrow) and islets are visible as small white dots (indicated with white arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52765/52765fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52765/52765fig4.jpg" /> 
Figure 4. PDL induces acinar apoptosis. Immunostaining for cleaved-Caspase 3 in PDL tail at 3, 7 and 14 days post-surgery reveals apoptotic bodies in the acinar compartment at day 3 and day 7 post surgery, while acinar cells are almost absent from PDL tail at day 14. Magnification bars are 25 &#181;m. Figure originally published by Xu et al., 2008.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52765/52765fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52765/52765fig5.jpg" /> 
Figure 5. PDL induces immune cell infiltration. Immunostaining for the leukocyte marker CD45, showing the presence of a high number of immune cells in PDL tail 7 days after surgery compared to sham pancreas. Magnification bars are 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52765/52765fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52765/52765fig6.jpg" /> 
Figure 6. PDL induces fibrosis. Immunostaining for alpha smooth muscle actin shows the presence of a fibrous network in PDL tail 14 days after surgery as compared to sham pancreas. Magnification bars are 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52765/52765fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52765/52765fig7.jpg" /> 
Figure 7. PDL induces an increase in duct cell proliferation. Double immunostaining for the duct cell marker Cytokeratin 19 (CK19) and for the proliferation marker BrdU shows an increase in cell cycle activity of duct cells. Magnification bars are 25 &#181;m. Figure originally published by Xu et al., 2008.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52765/52765fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Medicine

16.1K Views.  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. 

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

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Biology

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

Cancer Research

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Bioengineering

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 ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

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 ...

Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture

Source: Fleming Martinez, A. K. et. al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59096/" target="_blank">Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture</a>. J. Vis. Exp. (2019).
This video describes the technique of differentiating pancreatic acinar cells to ductal cells in ex vivo 3D culture. This helps to understand the mechanisms regulating the acinar-to-ductal metaplasia (ADM), a key process in the early development of pancreatic cancer.

Source: Fleming Martinez, A. K. et. al. Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture. J. Vis. Exp. ...

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

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