Name: minimizing post-infusion portal vein bleeding during intrahepatic islet transplantation in mice
Uploaded: 2021-05-10T15:00:00
Description: Watch this Scientific Journal Video about Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice at JoVE.com

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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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 ...

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