Hello, I'm me Doretta from the Diabetes Research Institute at the University of Miami Miller School of Medicine. I will demonstrate in this video how to transplant pancreatic eyelets into the interior chamber of the mouse eye. The procedure involves two steps.
Step one is loading the isolated pancreatic eyelets into the cannula, and step two is the actual transplantation procedure. For details about instrumentation and reagents, please refer to the accompanying text. We highlight here, the motorized syringe driver with the syringe mounted and the reservoir and cannula attachments, which will be focused on in the following video.
We begin by loading the eyelets into the cannula. All procedures are performed under the stereoscope. Begin by centering the eyelets by spinning or rotating the dish in circles.
When centered, disconnect the cannula from the reservoir and flush out any air bubbles by driving the motorized syringe driver forward. And that is done by stepping on the forward foot pedal. When ready, begin aspirating the eyelets into the reservoir by now driving the motorized syringe driver in the backward direction.
And again, that's done by stepping on the backward foot pedal. Notice how the eyelets tend to accumulate in the bottom of the reservoir. Then reconnect the cannula to the reservoir and eject the eyelets out of the reservoir.
Then the tubing into the cannula to dislodge the eyelets out of the reservoir and encourage them to stay packed in the tubing. Tap the reservoir as you drive the motorized syringe driver forward. Notice how packed the eyelets are in the tubing as they begin entering the back of the cannula.
By now, you should be ready to transplant the eyelets. Place the NS sized mouse on a pad and expose or pop the eye to be transplanted by pulling on the head skin with your index finger and thumb. Using a new needle as scalpel.
Approach the eye with the beveled end facing downward to prevent contact and injury of the iris. Penetrate the tip of the needle into the cornea and make a lateral incision. Next, introduce the cannula, preloaded with eyelids into the interior chamber through the incision, and begin infusing the eyelets into the chamber again by driving the motorized syringe driver forward.
Do not worry about air bubbles that might be trapped inside the cannula as they will dissipate later. And they will act as a natural barrier to minimize or prevent reflux of the eyelids out of the chamber. Slowly and carefully retract the cannula out of the interior chamber.
Now wash the eye with sterile PBS. Inject your preferred analgesic, and finally, apply ophthalmic antibiotic ointment. The transplantation is finished.
We now show representative results featuring pancreatic eyelids transplanted into the interior chamber of the eye. For longitudinal imaging studies of the individual eyelets on day seven. After transplantation, the eyelets are clearly engrafted on top of the iris by day 28.
The eyelets are in the same locations as seen previously 42 days after transplantation. The eyelets are clearly visible in the same locations. Also notice how well the incision healed precluding any possibility of interfering with imaging of the individual eyelets.
