I'm Thomas Linn, principle investigator of the Diabetes Research Group at Justus Liebig University, Giessen, Germany. Pancreatic islet transplantation is a treatment for type-1 diabetes mellitus. Experimental islet transplantation is performed to the kidney capsule, while clinical programs prefer the intra-portal route.
Therefore, we believe that more research on intra-portal islet transplantation is needed in preclinical models. For example, islets sites exposed to blood, as the transplantation site is intravascular. Moreover, the microsurgery technique needs some experience.
For example, after the injection of the portal vein, bleeding can occur, or there may be issues with anesthesia or with the maintenance of the bodyweight of the recipient mouse. Therefore, we think that visual explanation of the procedure is crucial. This video focuses on the intra-portal route of islet transplantation.
To achieve normal glycemia in diabetic mice, it can be used for syngeneic and allogenic transplantation in black mice, but also in syngeneic models in nude mice recipients. Remove the isolated islets from the incubator, and place them under a flow bench. Center the islets with a circular swinging motion of the petri dish.
Fill 100-microliter medium in one-ml syringe with the help of blue needle. Then add 350 islets in 300 microliter of Hank's Solution. Keep the syringe in a vertical position to allow the islets to settle.
Decrease the volume to 50 microliter. Replace the blue needle with the brown needle. To the same syringe add 150 microliter of density-gradient medium to the top, to reach to the final volume of 200 microliter in the syringe.
Avoid the air bubbles. Keep the syringe in vertical position. Rotate it slightly to avoid the clumping of islets.
The islets will settle at the cone of syringe within two to three minutes. After three days of streptozotocin injection, check the blood-glucose level by puncturing the tail vein with the help of handheld glucose meter. Anesthetize the mice with dalzylozine and ketamine by intraperitoneal injection.
Place the mice in supine position on warm thermal plate. Check the depth anesthesia by pinching the toe. Clean the skin with skinless infectant.
To prevent the eyes from drying, keep them moist with eye ointment. Start with electrotomy by holding the skin with forcep and making a two-to-three-centimeter-long middle incision with scissor. Place a sterile wet gauze around the wound edges to keep it moist.
Keep the intestine situs to the left and outside the abdominal cavity. Move the duodenum, and locate the pancreas. Use a finger and thumb to expose the portal vein.
Inject the islets in 200 microliter slowly into the portal vein. Apply the pressure with the index finger at the puncture site for six minute to stop the bleeding. Carefully place the intestine situs, peritoneum, and abdominal-muscles layer back into the position.
Using forcep, lift the skin, close the muscles incision, and upper-skin layer with absorbable silk suture. Apply a wound-healer. Maintain the body temperature at 37 degrees celsius on warm thermal plate.
Observe the mouse until it regains sufficient consciousness. Provide tramadol along with fluid to manage pain. After transplantation of islets, a sudden drop in blood-glucose level was observed on day one, from 23 millimolar per liter to 4.6 millimolar per liter in strepto-induced diabetic mice.
Normal glycemia was maintained for up to 121 days. The bodyweight and blood-glucose level roughly followed the same pattern. After the streptozotocin injection and surgery, the bodyweight decreased;from day two onwards it tend to rise towards normal, from 25.3 gram to 29.7 gram.
In major-histocompatibility-mismatch mice after islet transplantation, the blood-glucose level dropped to 4.4 millimole per liter, and the normal glycemia observed for up to 10 days. Afterward, it starts to reject. The bodyweight of diabetic mice also follows the similar pattern.
In successful engraftment of transplanted pancreatic islets into the liver. In this study, the intra-portal route of islet transplantation is explored. This protocol is highly efficient in relieving diabetic stress, and provide an in-depth opportunity to explore islet transplantation.
This protocol confirms that about 350 islets are capable of reversing the hyperglycemic state. Moreover, none of the mice died during the procedure, and the glycemic control was maintained.
