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...

<ol>
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L., 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=Activated+protein+C+preserves+functional+islet+mass+after+intraportal+transplantation:+A+novel+link+between+endothelial+cell+activation,+thrombosis,+inflammation,+and+islet+cell+death.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+tissue+factor+by+pancreatic+islet+cells+as+a+trigger+of+detrimental+thrombotic+reactions+in+clinical+islet+transplantation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrahepatic+islet+transplant+in+the+mouse:+functional+and+morphological+characterization.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+treatment+with+carbon+monoxide+can+yield+islet+allograft+survival+and+tolerance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+liver+histopathology+on+islet+cell+engraftment+in+the+model+mimicking+autologous+islet+cell+transplantation.">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/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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatoparenchymal+clearance+of+indocyanine+green+in+infectious+hepatitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+hepatocytes+and+sinusoidal+endothelial+cells+from+mouse+liver+perfusion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraportal+transplantation+of+pancreatic+islets+in+mouse+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+mesenchymal+stem+cell+and+islet+cotransplantation:+Safety+and+efficacy.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34; 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&#8221; 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&#160; 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 &#38; Upjohn Company</td>
			<td>34201</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe forceps 4&#8221; 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&#8221;</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...

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 ...

Liver is a primary site for transplantation in the human islet clinical setting. Mouse intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding. In this study, the two methods we showed both could effectively prevent bleeding-induced mouse death.Our method can be applied to research in liver cancer and other liver diseases by allowing injection of cancer cells, mesenchymal stem cells, and hepatocyte directly to the liver. To begin, detach a human islet culture with gentle scratching. Use a 1 cubic centimeter syringe to pick 300 to 350 islets into sterile 1.5 milliliter microcentrifuge tubes on ice.When all of the islets have been picked, centrifuge the tubes for 10 seconds to sediment the cells. Discard all but a small volume of supernatant to prevent pellet loss and resuspend the islets in 200 microliters HBSS containing 5%BSA. Load the resuspended islets into a 5 milliliter insulin syringe and turn the syringe needle-end up for 1 minute to let the islets settle at the plunger.Depress the plunger to remove any bubbles, keeping approximately 100 to 150 microliters of solution in the syringe and invert the syringe with gentle tapping to evenly distribute the islets. After confirming a lack of response to pedal reflex, apply ointment to the eyes to prevent drying. Shave the abdomen of the anesthetized mouse to remove the fur and disinfect the surgical area with three alternating wipes of 2%iodine and 75%alcohol.Next, make a 1 to 1.5-centimeter incision in the abdomen and open the peritoneal cavity. Place a sterile piece of gauze around the incision and use forceps or cotton tips to gently transfer the intestine onto the gauze. The then cover the intestine with a piece of saline-soaked gauze and expose the portal vein.To stop islet transfer-induced bleeding using gel foam, hold the needle with the bevel facing down and position the needle tip with the opening parallel to the portal vein wall before inserting the needle into the portal vein. Retract the plunger to draw 20 to 50 microliters of blood into the syringe to mix with the islets before infusing the islets into the portal vein while repeatedly depressing and retracting the plunger. When all of the islets have been delivered, place an approximately 5 by 5 centimeter piece of gel foam over the injection site.Use a cotton tip to press the gel foam down while removing the needle from the portal vein. Hold the gel in place for about 2 minutes, rolling the gel foam to make sure it covers the portal vein. Once the bleeding is stopped, gently return the intestine to the peritoneal cavity in the original position and inject 500 microliters of warm saline into the abdominal cavity.Use sutures to close the muscle and skin layers. Then place the mouse in a clean cage on a heating pad with monitoring until complete recovery from anesthesia, administering analgesia every 12 hours and heat for 48 hours. To stop islet transfer-induced bleeding using a fat pad, after exposing the portal vein as demonstrated, use two cotton tips to hold the exposed vein on the left and right sides and locate the fat tissue pad between the duodenum and portal vein.Penetrate the fat pad with the needle tip before insertion into the portal vein. Infuse the islets into the portal vein as demonstrated. When all of the islets have been delivered, press the fat tissue with a cotton tip for about 1 minute while removing the needle from the vein.Then return the intestine to the abdominal cavity and perform the rest of the postsurgical care procedures as demonstrated. The effects of the islet transplantation are dose-dependent, as normal glycemia is observed 30 days after syngeneic islet transplantation when 500 islets are transferred, but only transitory normoglycemia is observed when 250 islets are delivered, with the mice returning to hyperglycemia by 10 days post-transplant. An increased body weight is observed in both groups and identical responses observed when human islets are transplanted into diabetic NOD-SCID mice, with normoglycemia observed when higher numbers of islet equivalents are transplanted but not smaller numbers.The majority of the mice that have bleeding after the transfer die after the surgery, while the mice without bleeding survive. At 28 days post-transplantation, insulin staining reveals an even distribution of the human islet throughout the liver, mostly close to blood vessels. In addition to insulin, fibrin and polymorphonuclear cell filtration are also observed within the transplanted human islets.Intrahepatic islet transplantation will generate a large hole in the portal vein that may cause bleeding. Using our two improved procedures can prevent bleeding and reduce mortality post islet transplantation. The intraportal islet transplantation model can mimic clinical human islet transplantation.Our refined procedures can lead to higher survival rates for recipients, which allow further islet function study after transplantation.

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3.5K visualizações.  Medical University of South Carolina. O fígado é um local primário para transplante no cenário clínico da ilhota humana. O transplante de ilhotas intrahepticas de camundongos é tecnicamente desafiador, e uma alta porcentagem de camundongos pode morrer de complicações cirúrgicas, especialmente sangramento. Neste estudo, os dois métodos que mostramos ambos poderiam efetivamente prevenir a morte do camundongo induzida por sangramento.Nosso método pode ser aplicado a pesquisas em câncer de fígado e outras doenças...