Murine Model of Islet Transplantation: A Surgical Method to Infuse Pancreatic Islets in Portal Vein and Prevention of Postoperative Bleeding

Overview

In this video, we describe a procedure to infuse pancreatic islet cells into the hepatic portal vein, followed by application of gel foam to successfully prevent bleeding. Postoperative bleeding is a common cause of high mortality during islet transplantation.

Protocol

1. Islet transplantation

1. 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.
2. Shave and remove the fur in the abdomen area of the mouse.
3. Administer a single pre-operative dose of Buprenorphine (0.1 mg/kg i.p.).
4. Disinfect the surgical area with three alternating wipes of 2% iodine and 75% alcohol.
5. Perform a laparotomy with micro scissors to generate a 1-1.5 cm incision.
6. Open the peritoneal cavity with a retractor. Follow with either method A or method B as detailed below.

 Method A: (stop bleeding with gel foam, Figure 1A)

1. Mouse preparation
   1. Place a sterile gauze around the incision.
   2. Gently pull out the intestine using a forceps and keep it on the gauze.
   3. Identify the portal vein by its location and expose it well.
   4. Cover the intestine with a warm saline-wet gauze during the entire surgery.
2. 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's angle parallel to the portal vein wall before penetrating through the wall.
   1. Pull the plunger to draw some blood (20-50 μL) into the syringe to mix the islets first.
   2. Infuse the islets into the portal vein slowly while repeatedly pulling and pushing the plunge.
   3. Place a piece of gel foam (about 0.5 cm x 0.5 cm in size) to cover the injection site.
   4. Press the gel foam down with a cotton tip while pulling out the needle from the portal vein.
   5. Continue pressing on the gel for about 2 min to confirm there is no active bleeding.
   6. Rollover the cotton tip over and away from the gel foam to make sure the gel foam covers the portal vein well.

Method B: (stop bleeding with fat pad, Figure 1C)

1. Expose the portal vein thoroughly.
   1. Use two cotton tips to hold the exposed portal vein from both the left and the right sides.
   2. Identify the fat tissue pad between the duodenum and the portal vein.
   3. Penetrate through the fat pad before inserting the needle into the portal vein (Figure 1D).
   4. Infuse the islets, following the similar procedure described above in Method A.
   5. Pull out the needle while pressing down on the fat with a cotton tip.
   6. Continue pressing on the fat pad for 1 min after removing the needle.
2. After confirming that there is no bleeding from the portal vein, gently return the intestine to the peritoneal cavity in its original position.
3. Leave 0.5 mL of warm saline (36-37 °C) in the abdominal cavity before closure.
   NOTE: Warm saline facilitates post-surgery intestine movement and recovery and prevents intestine necrosis.
4. Close the muscle layer with an 5-0 suture.
5. Close the skin layer with an 4-0 suture.
6. Place the mouse in a clean cage on a heating pad until fully recovered from anesthesia.
7. 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.
   ​NOTE: The islet transplantation procedure takes approximately 15-20 min to complete.

Representative Results

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).

Materials

Name	Company	Catalog Number	Comments
10% Neutral buffered formalin v/v	Fisher Scientific	23426796
1 mL Syringe with needle	AHS	AH01T
20 mL Syringe	BD	301031
25G x 5/8" hypodermic needles	BD	305122
Alcohol prep pads, sterile	Fisher Scientific	22-363-750
Animal Anesthesia system	VetEquip, Inc.	Inc. 901806
Buprenorphine hydrochloride, injection	Par Sterile Products LLC	NDC 42023-179-05
Ethanol absolute (200 proof), molecular biology grade	Fisher Scientific	BP2818500
Extra fine Micro Dissecting scissors 4” straight sharp	Roboz Surgical Instrument Co.	RS-5882
Gelfoam (absorbable gelatin sponge USP)	Pharmacia & Upjohn Company	34201
Graefe forceps 4” extra delicate tip	Roboz Surgical Instrument Co.	RS-5136
Heated pad	Amazon	B07HMKMBKM
Hegar-Baumgartner Needle Holder 5.25”	Roboz Surgical Instrument Co.	RS-7850
Insulin syringe with 27-gauge needle	BD	879588
Iodine prep pads	Fisher Scientific	19-027048
Isoflurane	Piramal Critical Care	NDC 66794-017-25
Polypropylene Suture 4-0	Med-Vet International	MV-8683
Polypropylene Suture 5-0	Med-Vet International	MV-8661
Sodium chloride,  0.9% intravenous solution	VWR	2B1322Q
Surgical drape, sterile	Med-Vet International	DR1826