This work was supported by grants #2008-1-596 and #UNI-0404/1956 of the &#34;Tiroler Wissenschaftsfonds (TWF)&#34; (https://www.tirol.gv.at/en/), and by grant #2013-042018 of the &#34;MUI-Start F&#246;rderungsprogramm&#34; of the Medical University Innsbruck.

In order to avoid alloimmune responses, and to strictly investigate ischemia reperfusion injury-related graft damage, a syngeneic donor-recipient pair should be used. In this protocol, male C57BL6 (H2b) 10-12-week-old mice weighing 26 to 28 g were used as size-matched donor-recipient pairs. All animals were housed in a barrier pathogen free facility and received human care in compliance with the &#34;Principals of Laboratory Animal Care&#34; formulated by the National Society for Medical Research and the &#34;Guide for the Care and Use of Laboratory Animals&#34; prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication no. 86-23, revised 1985). The Austrian Ministry of Education, Science, and Culture approved the experiments described in this manuscript (BMWF-66.011/0056-II/3b/2011).
1. Pancreas Procurement
<ol>
	<li>Anesthetize the donor animal with an intraperitoneal (i.p.) injection of xylazine (5 mg/kg body weight) and ketamine (100 mg/kg body weight) using a 27-gauge (G) needle.</li>
	<li>Shave the hair in the abdominal region using an electrical shaver, and fix the mouse on the operative field in a supine position with strands of tape.</li>
	<li>Scrub the operative field three times with chlorhexidine-soaked gauzes.</li>
	<li>Perform a midline abdominal incision with a bilateral subcostal extension using scissors. Gently exteriorize the viscera to the left with sterile cotton sticks, and wrap them in a moistened gauze. Lift the xyphoid cranially with a mosquito clamp to provide maximal exposure of the abdominal cavity for the following steps.</li>
	<li>Using a 19 G needle, inject 400 &#181;L of a sterile 1:4 heparin-sodium chloride solution for heparinisation into the inferior vena cava (IVC). After removing the needle, euthanize the animal by exsanguination transecting of the aorta.</li>
	<li>Dissect the abdominal aorta between the origin of the superior mesenteric artery and the right renal artery by gently separating the fibrous tissue using curved tip forceps. Undermine the aorta and tie it with an 8/0 silk ligature.</li>
	<li>Identify the hepatoduodenal ligament, which runs between the postpyloric duodenum and the liver hilum. Divide the bile duct below the entrance of the cystic duct after distal ligature, and gently dissect and transect the portal vein as distally as possible to have enough length for performing the anastomosis in the recipient.</li>
	<li>Using curved tip forceps, bluntly dissect the abdominal aorta starting from the previous ligature (see step 1.6). Undermine the periaortic tissue using curved tip forceps, tie it with an 8/0 silk ligature, and transect it with scissors.
	<ol>
		<li>Using bipolar forceps, coagulate all its lumbar branches and transect the aorta with scissors as closely as possible to the diaphragm, in order to provide enough length for vessel anastomosis. At last, transect the already tied aorta above the left renal artery.</li>
	</ol>
	</li>
	<li>Perfuse the pancreas with a 19 G syringe with 5 mL of 4 &#176;C histidine-tryptophan-ketoglutarate perfusion solution in an antegrade fashion via the abdominal aorta, until there is a clear effluent coming from the portal vein. Apply low pressure to avoid oedema formation. 
	NOTE: Perform Steps 1.5 to 1.9 in a fast and standardized fashion to avoid any bias by warm ischemic deterioration of the recovered graft.</li>
	<li>Replace the viscera into the peritoneal cavity using sterile cotton sticks.</li>
	<li>Using curved tip forceps, separate the pancreas stepwise starting from the postpyloric duodenum, and moving forward until reaching the ligament of Treitz. For these steps, identify avascular connective tissue areas between the pancreas and the duodenal wall.
	<ol>
		<li>Bluntly dissect these areas using curved tip forceps in order to isolate bridging vessels between the pancreas and the duodenum. Pass an 8/0 silk ligature around each isolated vascular structure and tie it.</li>
		<li>Finally, transect the vascular structure with scissors towards the duodenal wall. In the same fashion, use an 8/0 silk suture to separate the pancreas from the mesentery, the transverse colon, and the stomach. 
		NOTE: During this procedure, the choledocho-pancreatic duct is ligated.</li>
	</ol>
	</li>
	<li>Identify the gastrosplenic ligament running from the spleen to the stomach, and the short gastric branches, by lifting the stomach cranially and cutting it with scissors. Leave the spleen attached to the recovered graft. 
	NOTE: In order to minimize graft rewarming, irrigate the pancreas continuously using a 10 mL syringe with a 19 G needle with the cold histidine-tryptophan-ketoglutarate perfusion solution stored on ice.</li>
	<li>Finally, remove the pancreas from the donor site (Figure 1B) by grasping the spleen with forceps, and transfer it into the recipient.
	<ol>
		<li>Alternatively, to trigger severe ischemia reperfusion injury, store the graft in sterile, 4 &#176;C perfusion solution for 16 h before implanting it into the recipient animal.</li>
	</ol>
	</li>
</ol>
2. Recipient Preparation
<ol>
	<li>Anesthetize the recipient animal with an i.p. injection of xylazine (5 mg/kg body weight) and ketamine (100 mg/kg body weight) using a 27 G needle.</li>
	<li>Shave the right lateral cervical region using an electrical shaver, and place the mouse on the operative field in a supine position. Fix the mouse using strands of tape. Avoid overstretching the front limbs to avoid compromising respiration.</li>
	<li>Scrub the operative field three times using chlorhexidine-soaked gauzes.</li>
	<li>Make a right paramedian, slightly oblique skin incision from the jugular incision to the right mandible angle.</li>
	<li>Bluntly identify and mobilize the lateral branches of the right external jugular vein. Coagulate them with bipolar forceps and transect them with scissors.</li>
	<li>Lift the right lobe of the submandibular salivary gland cranially, identify the vascular pedicle, and cauterize it using bipolar forceps. Remove the lobe by transecting the cauterized pedicle with scissors.</li>
	<li>In analogy to step 2.5, identify all medial branches of the external jugular vein, cauterize them with bipolar forceps and transect them with scissors. Undermine the external jugular vein with curved tip forceps as cranially as possible, and ligate it with two 8/0 silk ligatures, leaving enough space between the ligatures.</li>
	<li>Transect the external jugular vein between the two previously placed ligatures with straight scissors.</li>
	<li>Pass the proximal end of the external jugular vein through the polyethylene cuff (inner diameter of 0.75 mm, outer diameter of 0.94 mm), and fix the handle of the cuff with a venous microhemostat clamp.</li>
	<li>Remove the tie at the end of the vessel stump and evert the vessel over the cuff. Fix the everted vein on the cuff with a circular 8/0 - silk ligature (Figure 1A).</li>
	<li>Proximally and distally cauterize the superficial part of the right sternocleidomastoid muscle, transect it with scissors, and remove it.</li>
	<li>Gently mobilize the common carotid artery, undermine it below the bifurcation, and ligate it twice, making sure not to tie the carotid bifurcation. Cut the vessel between the ties.</li>
	<li>Similar to step 2.9, pass the proximal end of the common carotid artery through the polyamide cuff (inner diameter of 0.57 mm, outside diameter of 0.6 mm), and fix it with an arterial microvascular clamp.</li>
	<li>Remove the ligature at the end of the vascular stump, and gently widen the lumen using vessel dilatators. Evert the vessel over the arterial cuff, and fix it with an 8/0 silk ligature (Figure 1A).</li>
</ol>
3. Implantation
<ol>
	<li>Place the graft into the recipient&#39;s neck region using the spleen as a handle, with the head oriented laterally, the tail including the spleen medially, and the vessel stumps ventro-caudally. Use cotton sticks to position the graft properly.</li>
	<li>Gently pull the portal vein of the pancreatic graft over the recipient animal&#39;s external jugular vein, which has been previously everted, and fixed on the appropriate cuff (see step 2.10). Fix it with a circular 8/0 silk ligature.</li>
	<li>Pull the stump of the abdominal aorta of the graft over the everted common carotid artery of the recipient animal. Fix it with a circumferential 8/0 silk ligature (Figure 1C).</li>
	<li>Identify the splenic vessels close to the hilum of the spleen, and undermine them with curved tip forceps. Tie them with 8/0 silk ligatures and transect the splenic vessels to remove the spleen. Finally, shorten the ties.</li>
	<li>Using a clamp applying forceps, remove the clamp on the venous cuff first. Then remove the arterial clamp. 
	NOTE: If transplantation was successful, the pancreatic graft will be reperfused immediately showing a homogeneous pinkish color and visible arterial pulsation (Figure 1D).</li>
	<li>Moisten the graft with normothermic saline solution.</li>
	<li>Remove the handle of the venous cuff using straight forceps.</li>
	<li>Close the surgical wound with a running 6/0 suture.</li>
</ol>
4. Postoperative Care (Endpoint)
<ol>
	<li>Following the procedure, apply up to 0.5 mL of normal saline subcutaneously (s.c.) for replacement of intraoperative fluid loss using a 19 G needle.</li>
	<li>Keep the recipient animal on a heating pad until complete recovery from anaesthesia.</li>
	<li>Once awake, return the recipient animal to the housing facility, where it can have food and water ad libitum.</li>
	<li>To prevent postoperative pain, administer right after the operation (1) Buprenorphin (0.1 mg/kg b.w.) every 12 h for the first 5 days and (2) carprofen 4 mg/kg b.w. every 12 h s.c. for the first week.</li>
	<li>In order to estimate proper nutritional intake, monitor weight (g) of each recipient animal every day. A weight loss of more than 10-15% compared to weight at surgery day, apathy, crippling, a very bent back, as well as surgical side infections represent endpoints.
	<ol>
		<li>In this case, as well as after reaching clinical endpoint, sacrifice the animal using terminal isoflurane inhalation.</li>
	</ol>
	</li>
</ol>

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Y., Xue, L., Zheng, X., Yan, S., Zheng, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+transplantation+in+the+mouse.">Pancreas transplantation in the mouse.</a> Hepatobiliary Pancreat Dis Int. 9 (3), 254-258 (2010).</li><li>Maglione, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+pretreatment+with+tetrahydrobiopterin+saves+pancreatic+isografts+from+ischemia+reperfusion+injury+in+a+mouse+model.">Donor pretreatment with tetrahydrobiopterin saves pancreatic isografts from ischemia reperfusion injury in a mouse model.</a> Am J Transplant. 10 (10), 2231-2240 (2010).</li><li>Drognitz, O., Obermaier, R., von Dobschuetz, E., Pisarski, P., Neeff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+transplantation+and+ischemia-reperfusion+injury:+current+considerations.">Pancreas transplantation and ischemia-reperfusion injury: current considerations.</a> Pancreas. 38 (2), 226-227 (2009).</li><li>White, S., Shaw, J., Sutherland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+transplantation.">Pancreas transplantation.</a> Lancet. 373 (9677), 1808-1817 (2009).</li><li>Morath, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+pancreas-kidney+transplantation+in+type+1+diabetes.">Simultaneous pancreas-kidney transplantation in type 1 diabetes.</a> Clin Transplant. 23 (Suppl 21), 115-120 (2009).</li><li>Perseghin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-sectional+assessment+of+the+effect+of+kidney+and+kidney-pancreas+transplantation+on+resting+left+ventricular+energy+metabolism+in+type+1+diabetic-uremic+patients:+a+phosphorous-31+magnetic+resonance+spectroscopy+study.">Cross-sectional assessment of the effect of kidney and kidney-pancreas transplantation on resting left ventricular energy metabolism in type 1 diabetic-uremic patients: a phosphorous-31 magnetic resonance spectroscopy study.</a> J Am Coll Cardiol. 46 (6), 1085-1092 (2005).</li><li>Secchi, A., Caldara, R., La Rocca, E., Fiorina, P., Di Carlo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+disease+and+neoplasms+after+pancreas+transplantation.">Cardiovascular disease and neoplasms after pancreas transplantation.</a> Lancet. 352 (9121), 65 (1998).</li><li>Kirk, A. 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The authors declare that they have no competing financial interests.

IRI-associated graft damage is inherent to solid organ transplantation, and it is characterized by a disturbance of the microcirculation. Accumulation of several metabolites during the ischemic phase, and initiation of inflammatory cascades mediated mainly by reactive oxygen and nitrogen species, results in tissue damage during graft reperfusion4. This cascade may jeopardize not only short-term, but also long-term success and, hence, significantly influences patients survival14. To date, combined kidney pancreas transplantation represents the therapy of choice for patients suffering from type 1 diabetes with end stage renal disease15. Several studies have shown that a successful combined kidney pancreas transplantation does not only restore and protect kidney graft function in diabetic recipients, but also stabilizes or even reverses secondary complications, including neuropathy as well as micro- and macroangiopathy16,17,18.
Despite continuous efforts in Reduction, Replacement, and Refinement (3 R&#39;s) in animal research, reproduction of complex pathophysiological processes like IRI is merely impossible in in vitro settings. Therefore, animal models are still considered to be the ideal tool for translational research19,20. Mouse models like the one described here have several advantages compared to rat or other animal models. These include the availability of a vast quantity of genetically well-defined inbred mouse strains (e.g. transgenic and knock-out strains), a plethora of molecular analysis tools, as well as an easy and cheap handling21. A major advantage of the described model lies in the non-suture cuff technique. By using the herein presented technique, success rates of &#62;90% are achievable, which is dramatically better compared to previously described models22. Using this non-suture technique, we significantly reduced common complications like hypovolemic shock, thrombosis, and stenosis of the anastomoses12. A further advantage of this method consists of the extra abdominal position of the graft, which is associated with rapid postoperative recovery of the recipient. Additionally, the cervical location makes it perfectly suitable for in vivo analyses, such as live imaging of the graft by exterioration without any tension22.
The main drawback of this model is the occlusion of the pancreatic duct, which does not resemble clinical reality. In this model, exocrine drainage is managed by tying the choledocho-pancreatic duct. In the long term, this results in a marked fibrosis and atrophy of the gland without leading to graft pancreatitis22. Due to this deterioration of the exocrine tissue, which we observed as early as at day 30 after transplantation, we believe that this model is not suited for long term observation. In contrast, the unimpaired endocrine function makes gylcemic controls of the recipient an easy tool for daily assessment of the function of the graft13,23,24.
These characteristics makes this an ideal model for analyzing early graft injuries associated with long preservation periods or with different preservation solutions and techniques. To achieve optimal success with this model, several crucial steps must be considered. The pancreas itself is very susceptible to manipulation. Therefore, gentle handling using cotton sticks during organ recovery and during implantation minimizes mechanical trauma. Direct grasping of the gland with forceps should be avoided, since it would inevitably result in severe graft damage. For the same reason, the spleen is recovered together with the pancreas, and is used as a handle. This is also established in clinical practice. A further pitfall involves cold perfusion, which is achieved by perfusion via the aortic stump by using 4 &#176;C histidine-tryptophan-ketoglutarate perfusion solution. Hereby, an excessive swelling of the gland can be avoided by gently perfusing the graft. The remaining perfusion solution should be used for moistening the graft, in order to keep its temperature low during organ recovery.
With regard to recipient preparation a careful dissection of both the external jugular vein as well as the common carotid artery sets the base for successful revascularisation. In particular, complete exposure of the vein by removing not only all tributaries, but also the surrounding fat tissue, is necessary in order to avoid external compression and stenosis by remaining fat tissue. The selection of the appropriate cuff diameters is crucial. Based on shared experience, for mice weighing between 25 to 28 g, an inner diameter of 0.57 mm for the arterial cuff, and between 0.75 and 0.8 mm for the venous cuff, is appropriate. Precise, clean cutting of the edges of the cuffs is mandatory to avoid tearing the vessel stump. Dilatation of the vessels, especially of the artery, is achieved best by using vessel dilatators with fine tips. As a rule of thumb, the vessel should be able to widen to twice the lumen of the cuff. During the process of everting the vessel over and fixing it on the cuff, we recommend stabilizing vascular clamps by placing them under a skin flap, as this eases this crucial step.
As already mentioned, the non-suture cuff-technique represents an easy method for vascular anastomosis and can be performed within 5 min. However, correct positioning of the graft in the recipient&#39;s neck region is of utmost importance for correct revascularization. Hereby, the final correct positioning of the graft in the neck region has to be anticipated in order to allow a safe, straight, and tension-free anastomosis of both the vein and the artery. Vessels that are too long have to be avoided, since this may lead to outflow obstruction due to kinking. For the same reason, the cuff-handle at the venous anastomosis should also be removed following reperfusion. In cases of localized bleedings from the pancreatic graft, successful hemostasis can be achieved by gently compressing the bleeding side for 5 min using cotton sticks. This is the only successful way to manage this kind of complication. Cauterization, even though highly selective, resulted in graft loss in almost all cases, due to necrotic pancreatitis.
In summary, we developed a method for pancreas transplantation in mice using a non-suture cuff technique, which is technically and microsurgically feasible and has excellent success rates. Given the progredient fibrosis of the pancreas due to the duct occlusion, this model is suited best for research areas focusing on early graft damages. This manuscript is intended to allow researchers to safely establish this model in their laboratories.

Simultaneous kidney-pancreas transplantation (SPK) represents the current standard of care for patients suffering from diabetes mellitus and end stage renal disease. Successful transplantation results in long-term insulin independence associated with stabilization or even regression of diabetic microangiopathy and a better quality of life1. However, in contrast to other common solid organ transplantations, like kidney and liver transplantation, pancreatic grafts are more susceptible to ischemia reperfusion injury (IRI). Reported incidences of up to 35% jeopardize not only graft, but even patient, survival2,3.
Oxidative stress, microcirculatory disorders, increased expression of pro-inflammatory cytokines, and adhesion molecules resulting finally in endothelial activation and loss of its integrity, have all been attributed to this non-allogeneic graft injury4. So far, the exact molecular mechanisms of IRI are largely unknown and may vary from organ to organ.
Despite major progress utilizing in vitro models, the development of animal models is crucial to deepen the knowledge of molecular mechanisms involved in IRI-associated graft alterations following pancreas transplantation. Several pancreas transplantation models have been developed in rodents5,6, but only one is reported in mice7. The Achilles heel of this highly demanding microsurgical procedure is the low survival rate of 46%. However, mouse models represent the best model for transplant-related research, since the widest variety of molecular analysis tools can be applied to them. Based on extensive microsurgical experience in mice with different organ transplantations8,9,10, we developed a new, highly reproducible technique for heterotopic, cervical pancreas transplantation in mice with &#62;90% success rate using a non-suture cuff technique. With this technique, anastomoses-related complications are reduced to a minimum, and a high success rate can be achieved compared to the suture model11. So far, only one mouse model with similar success rates has been described by Liu et al12. However, there are no studies published using this model so far.

<table><tbody><tr><td>Adventitia Scissors</td><td>S&amp;amp;T</td><td>S-00102</td><td>Straight</td></tr><tr><td>Dumont # 7 Forceps</td><td>FST</td><td>&amp;nbsp;11271- 30</td><td>Curved Tip 0.17 x 0.1 mm</td></tr><tr><td>Yasargil Clip Mini Permanent 7mm</td><td>Aesculap</td><td>FE720K</td><td></td></tr><tr><td>Micro vessel clip</td><td>S&amp;amp;T</td><td>B1 00396 V</td><td></td></tr><tr><td>Vessel dilatator</td><td>S&amp;amp;T</td><td>D-5a.2, 00125</td><td></td></tr><tr><td>Clip applier</td><td>S &amp;amp; T</td><td>CAF-4 00072</td><td>for venous cuff</td></tr><tr><td>Clip applier</td><td>Aesculap</td><td>FE572K&amp;nbsp;</td><td>for the arterial cuff</td></tr><tr><td>Polyethylene tube</td><td>Portex Ltd</td><td></td><td>Inner diameter 0.75 mm for venous cuff</td></tr><tr><td>Polymide tubing</td><td>Vention Medical&amp;nbsp;</td><td>141-0051</td><td>Inner diameter 0.8 mm (Alternative for polyethylene tube from Portex Ltd)</td></tr><tr><td>Polymide tubing</td><td>Vention Medical</td><td>141-0033</td><td>Inner diameter 0.57 mm for arteriail cuff</td></tr><tr><td>Bipolar forceps</td><td>Micromed</td><td>140-100-015</td><td></td></tr><tr><td>8/0 silk ligatures</td><td>Catgut GmbH, Merkuramed</td><td>17209008</td><td></td></tr><tr><td>Custodiol HTK solution</td><td>Dr. Franz K&amp;ouml;hler Chemie</td><td>59997</td><td></td></tr><tr><td>Ketamin Graeub</td><td>aniMedica GmbH</td><td>32554</td><td></td></tr><tr><td>Xylasol Graeub</td><td>aniMedica GmbH</td><td>50855</td><td></td></tr></tbody></table>

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.

Over the last decade, we performed more than 300 pancreas transplantations in mice. After establishing the protocol, there was an overall survival of &#62;90%. Postoperative bleeding was the main cause for failure, followed by graft thrombosis with subsequent lethal necrotic graft pancreatitis. In both cases, endpoints were reached within 24 h and animals were sacrificed. There were not any neurological disorders, symptoms such as dysphagia, and surgical side infections in this series.
To investigate the endocrine function of the transplanted grafts, and hence validate the patency of the model, hyperglycemia was induced in recipient mice by pre-treatment with a single dose of intraperitoneally applied streptozotocin (312.5 mg/kg body weight) 4 days before surgery. Mice were considered hyperglycemic if blood glucose levels were &#62;300 mg/dL. Figure 2A shows the blood glucose levels of different groups. Mice receiving grafts without a prolonged cold ischemia time of 16 h reached normogylcemia within 24 h following transplantation, and maintained this metabolic state over the entire observation period. In contrast, non-transplanted animals remained hyperglycemic. Since we were interested in the impact of ischemia reperfusion injury-associated graft damage on endocrine function, we added a third group where grafts were exposed to 16 h prolonged cold ischemia time (CIT) and 45 min of warm ischemia time (WIT). Mice receiving these grafts did not reach normoglycemia and had to be sacrificed after 48 h due to the development of severe pancreatitis, which was shown to be lethal in this model13.
This model is useful for various projects aimed at investigating ischemia reperfusion injury-associated early graft damage. Further investigations included, among others, confocal intravital fluorescence microscopy for quantification of microcirculatory derangements performed 2 h following transplantation. Contrast of the grafts&#39; microvessels was enhanced by injecting 0.3 mL of a 0.4% fluorescein-isothiocyanate-labeled dextran (MW 150 000) into the penile vein. Figure 2B displays a regular capillary pattern of a na&#239;ve murine pancreas and of a transplanted pancreatic graft, which was not exposed to prolonged CIT (Figure 2C). In contrast, Figure 2D shows the breakdown of the microcirculation as a result of exposing the pancreatic graft to prolonged CIT.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54998/54998fig1.jpg" /> 
Figure 1: Intraoperative pictures. (A) Intraoperative view of the recipient vessels prepared for anastomosis. The external jugular vein (1) has been everted over the venous polyethylene cuff and fixed with a circular 8/0 silk ligature. In analogy, the common carotid artery (2) has been everted and fixed over the smaller arterial polyamide cuff. Scale bar 1 mm. (B) The pancreatic graft ex situ. The portal vein (1) and the stump of the abdominal aorta (2) needed for vascular anastomosis. The spleen (3) is retrieved together with the pancreas and is used as a handle. The spleen will be removed before reperfusion of the graft. Scale bar 1 cm. (C) Intraoperative view of the anastomoses. The portal vein (1) is pulled over the cuff of the everted external jugular vein (2) and fixed with a circular 8/0 silk tie. Similarly, the aortic stump of the abdominal aorta (3) is pulled over the cuff of the everted common carotid artery (4). Scale bar 1 mm. (D) Intraoperative view of the perfused pancreatic graft following 5 min reperfusion: After removal of the vein, followed by the arterial clamp, a successfully transplanted pancreatic graft displays a homogenous pinkish color. The spleen has been removed before reperfusion (1: ligated splenic vessel). Scale bar 1 cm. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54998/54998fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54998/54998fig2.jpg" /> 
Figure 2: Endocrine function of the pancreatic graft and confocal in vivo fluorescence microscopy. Figure 2A shows a line chart with blood glucose levels of transplanted mice without CIT (n = 10, PTX w/o CIT, blue line), non-transplanted mice (n = 11, no PTX, red line), and mice receiving grafts exposed to prolonged CIT (PTX + 16 h CIT, n = 10, green line). All recipients were previously rendered hyperglycemic with 312.5 mg/kg b.w. streptozotocin i.p. While all recipients of grafts without CIT were able to survive the entire observation period (50 days) with intact endocrine function, non-transplanted mice remained hyperglycemic over the entire observation period. Mice receiving grafts exposed to 16 h CIT did not recover from hyperglycemia and had to be sacrificed 48 h after transplant surgery, due to weight loss of more than 10-15%. Mice surviving the entire observation period were sacrificed at day 50 following one last glycemia measurement. Microcirculation in transplanted grafts was assessed by confocal intravital fluorescence microscopy 2 h following transplantation. Na&#239;ve pancreas served as controls. Figure 2B shows a regular capillary pattern in the na&#239;ve pancreas. A regular capillary mesh is also seen in transplanted grafts not subjected to prolonged CIT (Figure 2C). In contrast, a breakdown of the microcirculation is observed in transplanted grafts exposed to prolonged CIT (Figure 2D). Scale bar 100 &#181;m. Data in the graph are expressed as &#177; Standard Deviation. PTX: pancreas transplantation; CIT: cold ischemia time; w/o: without <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54998/54998fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique at JoVE.com

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

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 ...

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Universidad San Sebastian

Research

JoVE Journal

Medicine

10.9K Views.  Medical University Innsbruck. 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.

Video: Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

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

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 ...

Cancer Research

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

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

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 ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models

Source: An, X. et al.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/v/57460/" target="_blank">Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry</a>. J. Vis. Exp. (2018)
The video explains a stepwise surgical method for orthotopic implantation of a tumor fragment from a cancerous donor to the pancreas of a non-cancerous recipient mice for development of pancreatic cancer. Orthotopically implanted PDAC could recapture the tissue microenvironment of human pancreatic cancers than conventional subcutaneous (SC) models more accurately.

Source: An, X. et al.&#160;Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry. ...