This study was supported by the Department of Veterans Affairs (VA-ORD BLR&#38;D Merit I01BX004536), and the National Institute of Health grants # 1R01DK105183, DK120394, DK118529, to HW. We would like to thank you Mr. Michael Lee and Ms. Lindsay Swaby for language editing

All procedures were conducted with the approval of the Institutional Animal Care and Use Committees at the Medical University of South Carolina and the Ralph H Johnson Medical Center in Charleston.
1. Diabetes induction using streptozotocin (STZ)
<ol>
	<li>Recipient mice preparation:
	<ol>
		<li>Weigh all mice individually.</li>
		<li>Check blood glucose levels from a tail vein blood sample using a glucometer.</li>
	</ol>
	</li>
	<li>STZ dose determination for three different scenarios:
	<ol>
		<li>For mice with fatty liver disease inject one dose of STZ [40 mg/kg/day, intraperitoneal (i.p.) injection]&#160;for 5 consecutive days.</li>
		<li>For NOD-SCID mice inject 125 mg/kg of STZ, single injection, i.p. after overnight fasting.</li>
		<li>For C57BL/6 mice inject 225 mg/kg of STZ, single injection, i.p.</li>
	</ol>
	</li>
	<li>Calculations for STZ (13.5 mg/mL): 
	NOTE: This calculation is for five C57BL/6 mice with body weights of 30 g:
	<ol>
		<li>Total body weights: 5 mice x 30 g/mouse = 150g</li>
		<li>STZ needed: 150 g x 225 mg/1000g STZ = 33.75 mg</li>
	</ol>
	</li>
	<li>STZ preparation:
	<ol>
		<li>Weigh the STZ following the pre-calculated dose.</li>
		<li>Transfer the weighed STZ powder into a 10 mL beaker on ice.</li>
		<li>Add 3 mL of sodium citrate solution to the beaker to dissolve the STZ.</li>
		<li>Mix well,&#160;filter sterilize through a 0.22-&#956;m pore, and use the STZ solution within 10 min of preparation.</li>
	</ol>
	</li>
	<li>STZ injection:
	<ol>
		<li>Load the desired amount of STZ solution (enough for one mouse) into 1 mL syringe.</li>
		<li>Perform intraperitoneal injection at the lower right quadrant of mouse abdomen.</li>
		<li>Observe mice for 5 min after injection and check for any signs of discomfort during this period of time before putting them back into the cages.</li>
		<li>Monitor blood glucose level from a tail vein blood sample using a glucometer daily after the STZ injection. 
		NOTE: In this experiment, mice are considered diabetic when non-fasting blood glucose is &#62; 350 mg/dL for two consecutive days.</li>
	</ol>
	</li>
</ol>
2. Islet preparation
NOTE: Human islets were cultured in CMRL-1066 media supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (P/S) at a density of 10,000 islet equivalent number (IEQ) per 100 mm cell culture dish9. Mouse islets were cultured in DMEM with 10% FBS and 1% P/S with the same density13. Male NOD-SCID and C57BL/7 mice between 6-10 weeks of ages were obtained from commercial sources.
<ol>
	<li>Detach cultured islets from cell culture dish by gentle scratching.</li>
	<li>Hand-pick desired numbers of islets (e.g., 300-350 islets) using a 1cc syringe and put them into sterile 1.5 mL microcentrifuge tubes on ice.</li>
	<li>Spin the tube for 10 seconds using the microcentrifuge.</li>
	<li>Remove the supernatant, leaving some liquid to avoid losing the pellet.</li>
	<li>Resuspend the pellet in 200 &#956;L of HBSS with 0.5% bovine serum albumin (BSA).</li>
	<li>Aspirate the resuspended islets into a 0.5 mL insulin syringe.</li>
	<li>Place the syringe in the upright position. Let the islets sink down for 1 min.</li>
	<li>Push the syringe to remove all the bubbles, leaving about 100-150 &#956;L of liquid containing islets.</li>
	<li>Place syringe head down and gently tap the side of the syringe to let the islets equally distribute throughout the liquid. Islets are now ready for injection.</li>
</ol>
3. Islet transplantation
<ol>
	<li>Induce and maintain the mouse under general anesthesia with 2% isoflurane. Check for the lack of pedal reflexes to ensure proper anesthetization of the animal.</li>
	<li>Shave and remove the fur in the abdomen area of the mouse.</li>
	<li>Administer a single pre-operative dose of Buprenorphine (0.1 mg/kg i.p.).</li>
	<li>Disinfect the surgical area with three alternating wipes of 2% iodine and 75% alcohol.</li>
	<li>Perform a laparotomy with micro scissors to generate a 1-1.5 cm incision.</li>
	<li>Open the peritoneal cavity with a retractor. Follow with either method A or method B as detailed below.</li>
</ol>
4. Method A: (stop bleeding with gel foam,&#160;&#160;Figure 1A)14,15,16
<ol>
	<li>Mouse preparation
	<ol>
		<li>Place a sterile gauze around the incision.</li>
		<li>Gently pull out the intestine using a forceps and keep it on the gauze.</li>
		<li>Identify the portal vein by its location and expose it well.</li>
		<li>Cover the intestine with a warm saline-wet gauze during the entire surgery.</li>
	</ol>
	</li>
	<li>Insert the islet preloaded insulin syringe needle through the portal vein near the duodenum (Figure 1B). To do so, hold the needle with the hole (bevel) facing down and position the opening surface&#39;s angle parallel to the portal vein wall before penetrating through the wall.
	<ol>
		<li>Pull the plunger to draw some blood (20-50 &#956;L) into the syringe to mix the islets first.</li>
		<li>Infuse the islets into the portal vein slowly while repeatedly pulling and pushing the plunge.</li>
		<li>Place a piece of gel foam (about 0.5 cm x 0.5 cm in size) to cover the injection site.</li>
		<li>Press the gel foam down with a cotton tip while pulling out the needle from the portal vein.</li>
		<li>Continue pressing on the gel for about 2 min to confirm there is no active bleeding.</li>
		<li>Rollover the cotton tip over and away from&#160;the gel foam to make sure the gel foam covers the portal vein well.</li>
	</ol>
	</li>
</ol>
5. Method B: (stop bleeding with fat pad,&#160;&#160;Figure 1C)17
<ol>
	<li>Expose the portal vein thoroughly.
	<ol>
		<li>Use two cotton tips to hold the exposed portal vein from both the left and the right sides.</li>
		<li>Identify the fat tissue pad between the duodenum and the portal vein.</li>
		<li>Penetrate through the fat pad before inserting the needle into the portal vein (Figure 1D).</li>
		<li>Infuse the islets, following the similar procedure described above in part 4.2.1 and 4.2.2 of Method A.</li>
		<li>Pull out the needle while pressing down on the fat with a cotton tip.</li>
		<li>Continue pressing on the fat pad for 1 min after removing the needle.</li>
	</ol>
	</li>
	<li>After confirming that there is no bleeding from the portal vein, gently return the intestine to the peritoneal cavity in its original position.</li>
	<li>Leave 0.5 mL of warm saline (36-37 &#176;C) in the abdominal cavity before closure. 
	NOTE: Warm saline facilitates post-surgery intestine movement and recovery and prevents intestine necrosis.</li>
	<li>Close the muscle layer with an 5-0 suture.</li>
	<li>Close the skin layer with an 4-0 suture.</li>
	<li>Place the mouse in a clean cage on a heating pad until fully recovered from anesthesia.</li>
	<li>Continue to provide an analgesic (e.g., buprenorphine 0.1 mg/kg i.p.) every 12 h and supplemental heat for 48 h post-surgery. 
	&#8203;NOTE: The islet transplantation procedure takes approximately 15-20 min to complete.</li>
</ol>
6. H&#38;E staining and photograph of whole liver section
<ol>
	<li>Liver perfusion
	<ol>
		<li>Put the mouse under anesthesia as described above in part 3.1.</li>
		<li>Carefully expose the portal vein and cut the inferior vena cava.</li>
		<li>Manually perfuse the liver using 20 mL of 10% paraformaldehyde via the portal vein for about 5 minutes, using a 20 mL syringe with 25G needle18. 
		NOTE: Liver perfusion can remove blood from liver tissue and improve liver fixation without disturbing the islet grafts.</li>
		<li>Dissect the perfused whole liver from other organs.</li>
		<li>Fix the perfused liver tissue in 10% paraformaldehyde for 24 h.</li>
		<li>Embed the tissue in paraffin.</li>
		<li>Cut tissue sections of 5 &#181;m thickness each and put them on a glass slide for staining.</li>
		<li>Perform H&#38;E, insulin, fibrin, and polymorphonuclear neutrophil (PMN) staining using standard methods15,16.</li>
		<li>Scan whole liver section under a microscope.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Pellegrini, S., Cantarelli, E., Sordi, V., Nano, R., Piemonti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+the+art+of+islet+transplantation+and+cell+therapy+in+type+1+diabetes.">The state of the art of islet transplantation and cell therapy in type 1 diabetes.</a> Acta Diabetology. 53 (5), 683-691 (2016).</li><li>Ballinger, W. F., Lacy, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+intact+pancreatic+islets+in+rats.">Transplantation of intact pancreatic islets in rats.</a> Surgery. 72 (2), 175-186 (1972).</li><li>Wright, J. R., Hauptfeld, V., Lacy, P. E., 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=Induction+of+Ia+antigen+expression+on+murine+islet+parenchymal+cells+does+not+diminish+islet+allograft+survival.">Induction of Ia antigen expression on murine islet parenchymal cells does not diminish islet allograft survival.</a> American Journal of Pathology. 134 (2), 237-242 (1989).</li><li>Toyofuku, A., 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=Natural+killer+T-cells+participate+in+rejection+of+islet+allografts+in+the+liver+of+mice.">Natural killer T-cells participate in rejection of islet allografts in the liver of mice.</a> Diabetes. 55 (1), 34-39 (2006).</li><li>Goss, J. A., Nakafusa, Y., Finke, E. H., Flye, M. W., Lacy, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+tolerance+to+islet+xenografts+in+a+concordant+rat-to-mouse+model.">Induction of tolerance to islet xenografts in a concordant rat-to-mouse model.</a> Diabetes. 43 (1), 16-23 (1994).</li><li>Hara, 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=A+mouse+model+for+studying+intrahepatic+islet+transplantation.">A mouse model for studying intrahepatic islet transplantation.</a> Transplantation. 78 (4), 615-618 (2004).</li><li>Shapiro, A. 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=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>Wang, J., 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=Alpha-1+antitrypsin+enhances+islet+engraftment+by+suppression+of+instant+blood-mediated+inflammatory+reaction.">Alpha-1 antitrypsin enhances islet engraftment by suppression of instant blood-mediated inflammatory reaction.</a> Diabetes. 66 (4), 970-980 (2017).</li><li>Gou, W., 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=Alpha-1+antitrypsin+suppresses+macrophage+activation+and+promotes+islet+graft+survival+after+intrahepatic+islet+transplantation.">Alpha-1 antitrypsin suppresses macrophage activation and promotes islet graft survival after intrahepatic islet transplantation.</a> American Journal of Transplantation. , (2020).</li><li>Contreras, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+formulations+of+thrombomodulin+increase+engraftment+after+intraportal+islet+transplantation.">Liposomal formulations of thrombomodulin increase engraftment after intraportal islet transplantation.</a> Cell Transplantation. 19 (11), 1359-1367 (2010).</li><li>Cui, W., 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=Thrombomodulin+improves+early+outcomes+after+intraportal+islet+transplantation.">Thrombomodulin improves early outcomes after intraportal islet transplantation.</a> American Journal of Transplantation. 9 (6), 1308-1316 (2009).</li><li>Proto, C., Grasso, G., Fassio, P. 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All authors declare that they do not have conflict of interest.

In this study, two improved procedures that can prevent bleeding and may reduce mouse mortality during mouse IIT have been demonstrated. This study enables researchers to visualize the islet transplantation model that is unique in studying the instant blood mediated inflammatory response after transplantation. The IIT model is a distinctive model for studying islet cell survival and hepatic ischemic injuries in response to islet transplantation19. Here, we refined the procedure based on previous studies and reduced early complication-induced mouse mortality. Both method A14,15,16&#160;and method B8,9 were used in multiple studies. We showed that islets distributed among the whole liver, and neutrophil infiltration and thrombosis typically associated with IIT were prominent in graft immediately after transplantation.
There are several key steps in mouse hepatic islet transplantation. Because both human and mouse islets can be as large as 200 &#956;m in size, a needle size of at least 27G must be used for transplantation to ensure the islet products&#39; smooth flow. However, this would generate a large hole in the portal vein that may cause bleeding after needle removal. By injecting islets via the correct angle and using a dental sponge to block the injection site or injection through the fat tissue, the chance of bleeding can be minimized, and mice have higher survival rates after transplantation. These steps may also help avoid liver warm ischemia-reperfusion injuries caused by blockage of portal vein blood flow when performing this procedure19. They can also reduce the damages to the liver and the intestines that may contribute to mouse mortality post-surgery.
There are also several limitations of the mouse intrahepatic islet transplantation model compared to the human islet transplantations setting. First, we cannot monitor mouse portal vein pressure during islet infusion as we do in clinic settings. Second, the volume that can be transplanted into the mice may not reflect the high amount of islet product transplanted into a human. Therefore, the extent of thrombosis may be different. Thirdly, mouse islet grafts after transplantation will be temporarily exposed to a hyperglycemic environment since no insulin will be given to mice, while in humans20, insulin would be given during the peri-transplantation period to reduce the stress of transplanted islets20. Nevertheless, the intrahepatic islet model offers a unique pre-clinical model that can be used to study human islet transplantation.

Intraportal islet transplantation (IIT) via the portal vein is the most commonly used method for human islet transplantation in clinical settings. The mouse IIT model offers a great opportunity to study islet transplantation and test promising interventional approaches that can enhance the efficacy of islet transplantation1. IIT was first described in the 1970s and used by several groups1,2,3,4,5. It regained popularity after the breakthrough in human islet transplantation in the year 20006,7. However, most islet transplant studies used the kidney capsule as a preferred site for experimental islet transplantation due to its easy success. On the contrary, IIT is more technically challenging and less frequently used for islet transplantation studies8,9. Unlike IIT, however, islets transplanted under the kidney capsule do not suffer from the immediate blood-mediated inflammatory reaction characterized by thrombosis, inflammation, and hepatic tissue ischemia, and thus have better function than islets transplanted into the liver. The kidney capsule model, therefore, may not fully mimic the stresses encountered by islets in human islet transplantation10,11,12.
One of the major complications of IIT in mice is bleeding from the injection site after transplantation, which could cause 10-30% of mortality among different mouse strains12. In this paper, two refined approaches have been developed to stop bleeding more rapidly and securely and to reduce mouse mortality after an IIT. Visual demonstration of these refined details will help researchers identify the key steps of this technically challenging procedure.&#160;In addition, the location of the islet grafts in the recipient&#8217;s liver was determined by histological examination of the Hematoxylin and Eosin (H&#38;E) stained liver tissue (whole section) bearing transplanted islets.

<table><tbody><tr><td>10% Neutral buffered formalin v/v</td><td>Fisher Scientific</td><td>23426796</td><td></td></tr><tr><td>1 mL Syringe with needle</td><td>AHS</td><td>AH01T</td><td></td></tr><tr><td>20 mL Syringe</td><td>BD</td><td>301031</td><td></td></tr><tr><td>25G x 5/8&quot; hypodermic needles</td><td>BD</td><td>305122</td><td></td></tr><tr><td>Alcohol prep pads, sterile</td><td>Fisher Scientific</td><td>22-363-750</td><td></td></tr><tr><td>Animal Anesthesia system</td><td>VetEquip, Inc.</td><td>901806</td><td></td></tr><tr><td>Buprenorphine hydrochloride, injection</td><td>Par Sterile Products, LLC</td><td>NDC 42023-179-05</td><td></td></tr><tr><td>Centrifuge tubes, 15 mL</td><td>Fisher Scientific</td><td>0553859A</td><td></td></tr><tr><td>CMRL-1066</td><td>Corning</td><td>15110CV</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10013CV</td><td></td></tr><tr><td>Ethanol, absolute (200 proof), molecular biology grade</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>Extra fine Micro Dissecting scissors 4&amp;rdquo; straight sharp</td><td>Roboz Surgical Instrument Co.</td><td>RS-5882</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Corning</td><td>35011CV</td><td></td></tr><tr><td>FreeStyle&amp;nbsp; Glucose meter</td><td>Abbott</td><td>Lite</td><td></td></tr><tr><td>FreeStyle Blood Glucose test strips</td><td>Abbott</td><td>Lite</td><td></td></tr><tr><td>Gelfoam (absorbable gelatin sponge, USP)</td><td>Pharmacia &amp;amp; Upjohn Company</td><td>34201</td><td></td></tr><tr><td>Graefe forceps 4&amp;rdquo; extra delicate tip</td><td>Roboz Surgical Instrument Co.</td><td>RS-5136</td><td></td></tr><tr><td>Heated pad</td><td>Amazon</td><td>B07HMKMBKM</td><td></td></tr><tr><td>Hegar-Baumgartner Needle Holder 5.25&amp;rdquo;</td><td>Roboz Surgical Instrument Co.</td><td>RS-7850</td><td></td></tr><tr><td>Insulin syringe with 27-gauge needle</td><td>BD</td><td>879588</td><td></td></tr><tr><td>Iodine prep pads</td><td>Fisher Scientific</td><td>19-027048</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Critical Care</td><td>NDC 66794-017-25</td><td></td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>HyClone</td><td>SV30010</td><td></td></tr><tr><td>Polypropylene Suture 4-0</td><td>Med-Vet International</td><td>MV-8683</td><td></td></tr><tr><td>Polypropylene Suture 5-0</td><td>Med-Vet International</td><td>MV-8661</td><td></td></tr><tr><td>Sodium chloride, 0.9% intravenous solution</td><td>VWR</td><td>2B1322Q</td><td></td></tr><tr><td>Streptozocin (STZ)</td><td>Sigma</td><td>S0130</td><td></td></tr><tr><td>Surgical drape, sterile</td><td>Med-Vet International</td><td>DR1826</td><td></td></tr><tr><td>Tissue Cassette</td><td>Fisher Scientific</td><td>22-272416</td><td></td></tr></tbody></table>

minimizing post-infusion portal vein bleeding during intrahepatic islet transplantation in mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

We performed syngeneic and xenogeneic islet transplantations via the portal vein. Islet graft function was observed in a dose-dependent manner in both islet transplantation models. In the syngeneic islet transplantation model using C57BL/6 mice, transplantation of 250 islets led to transitory normoglycemia before mice returned to hyperglycemia. Mice receiving 500 islets reached and maintained normoglycemia beyond 30 days after transplantation (Figure 2A). Mice in both groups showed increased body weights (Figure 2B).
Similarly, in the human islets to diabetic NOD-SCID mouse islet transplantation model, islet graft function was compared when 45, 85, or 140 IEQs/kg of body weights were transplanted. Normoglycemia could not be achieved when 45 IEQ/g (~225-275 islets/mouse) human islets were transplanted. When the islet number increased to 85 IEQ/g (~ 400-450 islets/mouse), 35.7% (10/28) of the recipients achieved normoglycemia (p =0.02 vs. 45 IEQ/g group) at day 60 post-transplantation. Furthermore, 83.3% (5/6) of the recipients who received 140 IEQ/g (~ 600-650 islets/mouse) of human islets reached normoglycemia (Figure 2C). In addition, majority mice who had bleeding died after surgery while mice without bleeding survived (Figure 2D).
Once enough human islets are engrafted to NOD-SCID recipients, their blood glucose levels can be well-controlled at the early-stage post-transplantation and well-maintained until the end of the study. The grafted islets can be easily identified by H&#38;E and insulin staining. At 28 days post-transplantation, transplanted human islets were distributed evenly throughout the whole liver, mostly around/close to a blood vessel (Figure 3).
The intrahepatic model was used to demonstrate instant blood mediated inflammatory reaction as seen in human islet transplantation. In our tissue section, we observed expression of insulin, and presence of fibrin and Polymorphonuclear leukocytes&#160;infiltration in transplanted islets (Figure 4A-D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62530/62530fig1v2.jpg" /> 
Figure 1: Illustration of intrahepatic islet transplantation procedures. (A, C). Schematics of key steps used in Method A and Method B. (B, D). Islets were injected directly via the portal vein (C) or indirectly via fat pat (D). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62530/62530fig1v2large.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/62530/62530fig02.jpg" /> 
Figure 2:&#160;Representative outcomes of intraportal islet transplantation. (A, B). Syngeneic mouse islet intraportal transplantation. Pancreatic islets (250 or 500) from C57BL/6 mice were transplanted into male C57BL/6 mice that were rendered diabetic by STZ. (A) Serial blood glucose levels were measured. Normoglycemia was defined as glucose levels &#60;200 mg/dL for &#62;2 consecutive days. (B) Increase in the recipients&#39; body weight was observed post islet transplantation. (C) Percentage of diabetic NOD-SCID mice reaching normoglycemia in mice receiving a different number of human islets at 45 IEQ/g (n=7), 85 IEQ/g (n=28), and 140 IEQ/g (n=6). (D) Percentage of survival after IIT in bleeding and non-bleeding mice (n=14 each). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62530/62530fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62530/62530fig3v5.jpg" /> 
Figure 3: H&#38;E staining of liver sections of NOD-SCID liver bearing human islet graft at 28 days post-transplantation. Islets are marked by black circles. The diameter of each circle positively corresponds with the size of each islet. Scale bar =1,000 &#956;m in whole liver section and 100 &#956;m in inset. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62530/62530fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62530/62530fig4v2.jpg" /> 
Figure 4: Representative histological pictures of intraportal transplanted mouse islets in liver 6 h after intraportal transplantation. (A) H&#38;E, (B) insulin (red) (C) Fibrin, and (D) PMN stains. Scale bar = 100 &#956;m.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62530/62530fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice at JoVE.com

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

minimizing-post-infusion-portal-vein-bleeding-during-intrahepatic

Universidad San Sebastian

Research

JoVE Journal

Medicine

3.6K Views.  Medical University of South Carolina. Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Video: Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

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 ...

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

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming increasingly common. Ex vivo liver perfusions allow for a comprehensive analysis of liver metabolism using nuclear magnetic resonance (NMR), in nutritional conditions that can be rigorously controlled. As in silico simulations remain a primarily theoretical means of assessing hormone actions and the effects of pharmaceutical intervention, the perfused liver remains one of the most valuable test beds for understanding hepatic metabolism. As these studies guide basic insights into hepatic physiology, results must be accurate and reproducible. The greatest factor in the reproducibility of ex vivo hepatic perfusion is the quality of surgery. Therefore, we have introduced an organized and streamlined method to perform ex vivo mouse liver perfusions in the context of in situ NMR experiments. We also describe a unique application and discuss common issues encountered in these studies. The overall purpose is to provide an uncomplicated guide to a technique we have refined over several years that we deem the golden standard for obtaining reproducible results in hepatic resections and perfusions in the context of in situ NMR experiments. The distance to the center of the field for the magnet as well as the inaccessibility of the tissue to intervention during the NMR experiment makes our methods novel.

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming ...

Biochemistry

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

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

Intraportal Transplantation of Pancreatic Islets in Mouse Model

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

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

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

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Biology

Mice Liver Perfusion for Accurate Evaluation of Hepatic Insulin Clearance

Assessing Insulin Clearance in Mice via In Situ Liver Perfusion

Hepatic insulin clearance is essential for maintaining glucose homeostasis and is closely linked to metabolic disorders such as obesity, insulin resistance, and diabetes. Accurate measurement of insulin clearance is vital for understanding the underlying mechanisms of these conditions. This protocol presents a straightforward and user-friendly hepatic perfusion procedure in mice, specifically designed to directly evaluate the hepatic insulin clearance rate. The method involves precise cannulation of the portal vein and suprahepatic inferior vena cava to create an in situ perfusion system that mimics physiological conditions. The protocol guides researchers through every stage of the procedure, from surgical preparation and setting up the perfusion system to sample collection and analysis. Detailed instructions are provided, along with representative results and important tips for optimizing the procedure. A video tutorial accompanies the written protocol, offering visually in-depth instructions and illustrations, making it an accessible and comprehensive reference for scientists exploring the molecular mechanisms behind hepatic insulin metabolism and clearance.

Hepatic insulin clearance is critical for regulating glucose homeostasis. This article describes a user-friendly hepatic perfusion procedure for directly evaluating the hepatic insulin clearance rate in situ in mice.

Hepatic insulin clearance is essential for maintaining glucose homeostasis and is closely linked to metabolic disorders such as obesity, insulin ...

Portal Vein Injection: A Method to Study Cancer Metastasis to the Liver

Source: Goddard, E. T., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/54903/a-portal-vein-injection-model-to-study-liver-metastasis-breast">A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer.</a> J. Vis. Exp. (2016).
This video describes a surgical procedure to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits the investigation of late stages of liver metastasis.

Source: Goddard, E. T., et al. A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer. J. Vis. Exp. (2016).
This video describes a ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Summary

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Chapters in this video

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

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

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

A Method for Islet Transplantation to the Omentum in Mouse

Assessing Insulin Clearance in Mice via In Situ Liver Perfusion

Portal Vein Injection: A Method to Study Cancer Metastasis to the Liver