Name: Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice (Video) | JoVE Journal
Uploaded: 2021-05-10T15:00:00.000+00:00
Duration: 6 min 41 s
Description: Learn refined surgical techniques for intraportal islet transplantation in mice using gel foam and fat pad methods. These procedures effectively prevent bleeding-induced mortality and advance liver disease research.

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5 minutes

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/pubmed?term=((The+state+of+the+art+of+islet+transplantation+and+cell+therapy+in+type+1+diabetes%5BTitle%5D)+AND+%22Acta+Diabetology%22%5BJournal%5D)">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/pubmed?term=((Transplantation+of+intact+pancreatic+islets+in+rats%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">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/pubmed?term=((Induction+of+Ia+antigen+expression+on+murine+islet+parenchymal+cells+does+not+diminish+islet+allograft+survival%5BTitle%5D)+AND+%22American+Journal+of+Pathology%22%5BJournal%5D)">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/pubmed?term=((Natural+killer+T-cells+participate+in+rejection+of+islet+allografts+in+the+liver+of+mice%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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/pubmed?term=((Induction+of+tolerance+to+islet+xenografts+in+a+concordant+rat-to-mouse+model%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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/pubmed?term=((A+mouse+model+for+studying+intrahepatic+islet+transplantation%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">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/pubmed?term=((Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">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/pubmed?term=((Alpha-1+antitrypsin+enhances+islet+engraftment+by+suppression+of+instant+blood-mediated+inflammatory+reaction%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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/pubmed?term=((Alpha-1+antitrypsin+suppresses+macrophage+activation+and+promotes+islet+graft+survival+after+intrahepatic+islet+transplantation%5BTitle%5D)+AND+%22American+Journal+of+Transplantation%22%5BJournal%5D)">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. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activated+protein+C+preserves+functional+islet+mass+after+intraportal+transplantation%3A+A+novel+link+between+endothelial+cell+activation%2C+thrombosis%2C+inflammation%2C+and+islet+cell+death%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Activated protein C preserves functional islet mass after intraportal transplantation: A novel link between endothelial cell activation, thrombosis, inflammation, and islet cell death.</a> Diabetes. 53 (11), 2804-2814 (2004).</li>
<li>Moberg, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+tissue+factor+by+pancreatic+islet+cells+as+a+trigger+of+detrimental+thrombotic+reactions+in+clinical+islet+transplantation%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation.</a> Lancet. 360 (9350), 2039-2045 (2002).</li>
<li>Melzi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrahepatic+islet+transplant+in+the+mouse%3A+functional+and+morphological+characterization%5BTitle%5D)+AND+%22Cell+Transplantation%22%5BJournal%5D)">Intrahepatic islet transplant in the mouse: functional and morphological characterization.</a> Cell Transplantation. 17 (12), 1361-1370 (2008).</li>
<li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Donor+treatment+with+carbon+monoxide+can+yield+islet+allograft+survival+and+tolerance%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Donor treatment with carbon monoxide can yield islet allograft survival and tolerance.</a> Diabetes. 54 (5), 1400-1406 (2005).</li>
<li>Desai, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+liver+histopathology+on+islet+cell+engraftment+in+the+model+mimicking+autologous+islet+cell+transplantation%5BTitle%5D)+AND+%22Islets%22%5BJournal%5D)">Effect of liver histopathology on islet cell engraftment in the model mimicking autologous islet cell transplantation.</a> Islets. 9 (6), 140-149 (2017).</li>
<li>Cui, W., Angsana, J., Wen, J., Chaikof, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liposomal+formulations+of+thrombomodulin+increase+engraftment+after+intraportal+islet+transplantation%5BTitle%5D)+AND+%22Cell+Transplantation%22%5BJournal%5D)">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/pubmed?term=((Thrombomodulin+improves+early+outcomes+after+intraportal+islet+transplantation%5BTitle%5D)+AND+%22American+Journal+of+Transplantation%22%5BJournal%5D)">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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatoparenchymal+clearance+of+indocyanine+green+in+infectious+hepatitis%5BTitle%5D)+AND+%22Giornale+di+Malattie+Infettive+e+Parassitarie%22%5BJournal%5D)">Hepatoparenchymal clearance of indocyanine green in infectious hepatitis.</a> Giornale di Malattie Infettive e Parassitarie. 20 (9), 845-851 (1968).</li>
<li>Cabral, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+hepatocytes+and+sinusoidal+endothelial+cells+from+mouse+liver+perfusion%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Purification of hepatocytes and sinusoidal endothelial cells from mouse liver perfusion.</a> Journal of Visualized Experiments. (132), e56993(2018).</li>
<li>Khatri, R., Hussmann, B., Rawat, D., Gurol, A. O., Linn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intraportal+transplantation+of+pancreatic+islets+in+mouse+model%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Intraportal transplantation of pancreatic islets in mouse model.</a> Journal of Visualized Experiments. (135), e57559(2018).</li>
<li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autologous+mesenchymal+stem+cell+and+islet+cotransplantation%3A+Safety+and+efficacy%5BTitle%5D)+AND+%22Stem+Cells+Translational+Medicine%22%5BJournal%5D)">Autologous mesenchymal stem cell and islet cotransplantation: Safety and efficacy.</a> Stem Cells Translational Medicine. 7 (1), 11-19 (2018).</li>
</ol>

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 s...

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

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...

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

Research

JoVE Journal

Medicine

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A Method for Islet Transplantation to the Omentum in Mouse

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.

Intraportal Transplantation of Pancreatic Islets in Mouse Model

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.

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

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.

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

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

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

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.

Assessing Insulin Clearance in Mice via In Situ Liver Perfusion

Here, we provide details on performing glucose-stimulated insulin secretion (GSIS) via perfusion through the mice vasculature with an intact pancreas as an additional technique to assess the endocrine pancreatic function with reduction of possible confounders.

Glucose-Stimulated Insulin Secretion via Perfusion through the Mice Vasculature with an Intact Pancreas

Learn refined surgical techniques for intraportal islet transplantation in mice using gel foam and fat pad methods. These procedures effectively prevent bleeding-induced mortality and advance liver disease research.

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

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