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>

<ol><li>Shaw, J. E., Sicree, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+estimates+of+the+prevalence+of+diabetes+for%5BTitle%5D)+AND+%22Diabetes+Res+Clin+Pract%22%5BJournal%5D)">Global estimates of the prevalence of diabetes for.</a> Diabetes Res Clin Pract. 87, 4-14 (2010).</li>
<li>Danaei, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((National%2C+regional%2C+and+global+trends+in+fasting+plasma+glucose+and+diabetes+prevalence+since+1980%3A+systematic+analysis+of+health+examination+surveys+and+epidemiological+studies+with+370+country-years+and+2%C2%B77+million+participants%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2·7 million participants.</a> Lancet. 378, 31-40 (2011).</li>
<li>Borowiak, M., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+make+beta+cells%5BTitle%5D)+AND+%22Current+opinion+in+Cell+Biology%22%5BJournal%5D)">How to make beta cells.</a> Current opinion in Cell Biology. 21, 727-732 (2009).</li>
<li>Gradwohl, G., Dierich, A., LeMeur, M., Guillemot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((neurogenin3+is+required+for+the+development+of+the+four+endocrine+cell+lineages+of+the+pancreas%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas.</a> Proc Natl Acad Sci U S A. 97, 1607-1611 (2000).</li>
<li>Gu, G., Dubauskaite, J., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+evidence+for+the+pancreatic+lineage%3A+NGN3%2B+cells+are+islet+progenitors+and+are+distinct+from+duct+progenitors%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.</a> Development. 129, 2447-2457 (2002).</li>
<li>Dor, Y., Brown, J., Martinez, O. I., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+pancreatic+beta-cells+are+formed+by+self-duplication+rather+than+stem-cell+differentiation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.</a> Nature. 429, 41-46 (2004).</li>
<li>Teta, M., Rankin, M. M., Long, S. Y., Stein, G. M., Kushner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+and+regeneration+of+adult+beta+cells+does+not+involve+specialized+progenitors%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Growth and regeneration of adult beta cells does not involve specialized progenitors.</a> Dev Cell. 12, 817-826 (2007).</li>
<li>Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perspective%3A+Postnatal+pancreatic+beta+cell+growth%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Perspective: Postnatal pancreatic beta cell growth.</a> Endocrinology. 141, 1926-1929 (2000).</li>
<li>Brennand, K., Huangfu, D., Melton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((All+beta+cells+contribute+equally+to+islet+growth+and+maintenance%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">All beta cells contribute equally to islet growth and maintenance.</a> PLoS Biol. 5, e163(2007).</li>
<li>Salpeter, S. J., Klein, A. M., Huangfu, D., Grimsby, J., Dor, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose+and+aging+control+the+quiescence+period+that+follows+pancreatic+beta+cell+replication%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Glucose and aging control the quiescence period that follows pancreatic beta cell replication.</a> Development. 137, 3205-3213 (2010).</li>
<li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beta+cells+can+be+generated+from+endogenous+progenitors+in+injured+adult+mouse+pancreas%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas.</a> Cell. 132, 197-207 (2008).</li>
<li>Wang, R. N., Kloppel, G., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Duct-+to+islet-cell+differentiation+and+islet+growth+in+the+pancreas+of+duct-ligated+adult+rats%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats.</a> Diabetologia. 38, 1405-1411 (1995).</li>
<li>Yasuda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokine+expression+and+induction+of+acinar+cell+apoptosis+after+pancreatic+duct+ligation+in+mice%5BTitle%5D)+AND+%22Journal+of+interferon%2C+%26+cytokine+research+%3A+the+official+journal+of+the+International+Society+for+Interferon+and+Cytokine+Research%22%5BJournal%5D)">Cytokine expression and induction of acinar cell apoptosis after pancreatic duct ligation in mice.</a> Journal of interferon, & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 19, 637-644 (1999).</li>
<li>Van Gassen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophage+dynamics+are+regulated+by+local+macrophage+proliferation+and+monocyte+recruitment+in+injured+pancreas%5BTitle%5D)+AND+%22European+journal+of+immunology%22%5BJournal%5D)">Macrophage dynamics are regulated by local macrophage proliferation and monocyte recruitment in injured pancreas.</a> European journal of immunology. , (2015).</li>
<li>Van de Casteele, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurogenin+3%2B+cells+contribute+to+beta-cell+neogenesis+and+proliferation+in+injured+adult+mouse+pancreas%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">Neurogenin 3+ cells contribute to beta-cell neogenesis and proliferation in injured adult mouse pancreas.</a> Cell Death Dis. 4, e523(2013).</li>
<li>Xiao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((No+evidence+for+beta+cell+neogenesis+in+murine+adult+pancreas%5BTitle%5D)+AND+%22The+Journal+of+clinical+investigation%22%5BJournal%5D)">No evidence for beta cell neogenesis in murine adult pancreas.</a> The Journal of clinical investigation. 123, 2207-2217 (2013).</li>
<li>Ackermann Misfeldt, A., Costa, R. H., Gannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beta-cell+proliferation%2C+but+not+neogenesis%2C+following+60%25+partial+pancreatectomy+is+impaired+in+the+absence+of+FoxM1%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Beta-cell proliferation, but not neogenesis, following 60% partial pancreatectomy is impaired in the absence of FoxM1.</a> Diabetes. 57, 3069-3077 (2008).</li>
<li>Lee, C. S., De Leon, D. D., Kaestner, K. H., Stoffers, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+of+pancreatic+islets+after+partial+pancreatectomy+in+mice+does+not+involve+the+reactivation+of+neurogenin-3%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Regeneration of pancreatic islets after partial pancreatectomy in mice does not involve the reactivation of neurogenin-3.</a> Diabetes. 55, 269-272 (2006).</li>
<li>Bonner-Weir, S., Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pancreatic+stem+cells%5BTitle%5D)+AND+%22The+Journal+of+pathology%22%5BJournal%5D)">Pancreatic stem cells.</a> The Journal of pathology. 197, 519-526 (2002).</li>
<li>Nir, T., Melton, D. A., Dor, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recovery+from+diabetes+in+mice+by+beta+cell+regeneration%5BTitle%5D)+AND+%22The+Journal+of+clinical+investigation%22%5BJournal%5D)">Recovery from diabetes in mice by beta cell regeneration.</a> The Journal of clinical investigation. 117, 2553-2561 (2007).</li>
<li>Bouwens, L., Rooman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+pancreatic+beta-cell+mass%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Regulation of pancreatic beta-cell mass.</a> Physiol Rev. 85, 1255-1270 (2005).</li>
<li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurog3+gene+dosage+regulates+allocation+of+endocrine+and+exocrine+cell+fates+in+the+developing+mouse+pancreas%5BTitle%5D)+AND+%22Developmental+biology%22%5BJournal%5D)">Neurog3 gene dosage regulates allocation of endocrine and exocrine cell fates in the developing mouse pancreas.</a> Developmental biology. 339, 26-37 (2010).</li>
<li>Pan, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+patterns+of+multipotentiality+in+Ptf1a-expressing+cells+during+pancreas+organogenesis+and+injury-induced+facultative+restoration%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Spatiotemporal patterns of multipotentiality in Ptf1a-expressing cells during pancreas organogenesis and injury-induced facultative restoration.</a> Development. 140, 751-764 (2013).</li>
<li>Kopp, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sox9%2B+ductal+cells+are+multipotent+progenitors+throughout+development+but+do+not+produce+new+endocrine+cells+in+the+normal+or+injured+adult+pancreas%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas.</a> Development. 138, 653-665 (2011).</li>
<li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sustained+Neurog3+expression+in+hormone-expressing+islet+cells+is+required+for+endocrine+maturation+and+function%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function.</a> Proc Natl Acad Sci U S A. 106, 9715-9720 (2009).</li>
<li>Kopinke, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lineage+tracing+reveals+the+dynamic+contribution+of+Hes1%2B+cells+to+the+developing+and+adult+pancreas%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas.</a> Development. 138, 431-441 (2011).</li>
<li>Rankin, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((beta-Cells+are+not+generated+in+pancreatic+duct+ligation-induced+injury+in+adult+mice%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">beta-Cells are not generated in pancreatic duct ligation-induced injury in adult mice.</a> Diabetes. 62, 1634-1645 (2013).</li>
<li>Solar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pancreatic+exocrine+duct+cells+give+rise+to+insulin-producing+beta+cells+during+embryogenesis+but+not+after+birth%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Pancreatic exocrine duct cells give rise to insulin-producing beta cells during embryogenesis but not after birth.</a> Dev Cell. 17, 849-860 (2009).</li>
<li>Furuyama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+cell+supply+from+a+Sox9-expressing+progenitor+zone+in+adult+liver%2C+exocrine+pancreas+and+intestine%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.</a> Nat Genet. 43, 34-41 (2011).</li>
<li>Van de Casteele, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Partial+duct+ligation%3A+beta-cell+proliferation+and+beyond%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Partial duct ligation: beta-cell proliferation and beyond.</a> Diabetes. 63, 2567-2577 (2014).</li>
<li>Zhang, J., Rouse, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histopathology+and+pathogenesis+of+caerulein-%2C+duct+ligation-%2C+and+arginine-induced+acute+pancreatitis+in+Sprague-Dawley+rats+and+C57BL6+mice%5BTitle%5D)+AND+%22Histology+and+histopathology%22%5BJournal%5D)">Histopathology and pathogenesis of caerulein-, duct ligation-, and arginine-induced acute pancreatitis in Sprague-Dawley rats and C57BL6 mice.</a> Histology and histopathology. 29, 1135-1152 (2014).</li>
<li>Martinez-Romero, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+epigenetic+regulators+Bmi1+and+Ring1B+are+differentially+regulated+in+pancreatitis+and+pancreatic+ductal+adenocarcinoma%5BTitle%5D)+AND+%22The+Journal+of+pathology%22%5BJournal%5D)">The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma.</a> The Journal of pathology. 219, 205-213 (2009).</li>
<li>Prevot, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+ductal+transcription+factors+HNF6+and+Sox9+in+pancreatic+acinar-to-ductal+metaplasia%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia.</a> Gut. 61, 1723-1732 (2012).</li>
</ol>

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><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 ...

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

16.2K 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

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

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 ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Developmental Biology

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

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

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 ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Biology

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

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

Summary

Explore More Videos

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

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

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

Lepr^db Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

A System for ex vivo Culturing of Embryonic Pancreas

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis