Grafting Pancreatic Islets: A Method to Implant Pancreatic Islets into the Anterior Chamber of the Mouse Eye for In Vivo Imaging
내레이션 대본
– Pancreatic islets contain beta cells that secrete insulin. In type-1 diabetes patients, insulin-making beta cells become dysfunctional. Transplantation of islets in such patients offers potential for providing glycemic control. To evaluate the transplantation efficiency, we use animal models such as a mouse whose anterior eye chamber is an optimal site for grafting and in vivo imaging.
For transplanting islets, place an anesthetized mouse on a warm heating pad and inject buprenorphine, an analgesic to relieve pain, subcutaneously at the back of the mouse. Next, insert the mouse's nose into a nose mask for a continuous supply of oxygen and anesthetic isoflurane.
Fix the mouse's head in the head holder to expose the eye and gently retract the eyelid using blunt forceps to pop the eye out. Load a cannula with fluorescently labeled islets. Insert the cannula's half tip into the cornea. Lift the cornea, gently dispense the islets, remove the cannula, and apply eye gel.
After the desired duration, place the mouse under a two-photon microscope and take images. You may observe blood vessels forming a microvascular network throughout the graft. In the following protocol, we will perform human pancreatic islet grafting into the mouse's anterior eye chamber.
To begin, place the mouse on a warm heating pad, position it in the head holder, and put on the nose mask. Use the thumb and index finger to lift the head slightly. Fasten it with the metallic pieces on the sides, making sure that the earpieces fix the head directly below the ears. Subcutaneously, inject the buprenorphine solution into the back of the mouse. Gently, retract the eyelids of the eye to be transplanted using blunt forceps. Pop the eye out and loosely fix it with a pair of tweezers covered with a polythene tube.
After transferring the islets to a Petri dish with PBS, pick up approximately 20 to 30 islets in the eye cannula. Using a 25-gauge needle, bevel upwards, carefully insert the tip into the cornea and make a single lateral incision. Carefully lift the cornea with the cannula preloaded with islets and slowly release the islets into the eye. Slowly retract the cannula and apply eye gel to the eye.
After 10 minutes, remove the forceps holding the eyelid and put the eye back to its normal position. For imaging of implanted human islets with two-photon microscopy, place the head holder platform under the microscope and administer eye gel onto the eye as an immersion liquid between the cornea and the lens. Position the eye under the objective and image the implanted human islets as described in the text manuscript.
