The overall goal of these procedures is to separate the influence of reproductive aging from chronological aging in females. This method can help answer key questions in the biomedical field such as how do ovarian signals from young ovaries preserve healthy young females, and restore health in menopausal females? The main advantage of this technique is that we can transfer components of ovarian signaling from a young healthy animal to an older animal that has lost its young signaling.
The implications of this technique extend toward the therapy of aging associated disease. As many of these diseases can be avoided in old mice through the restoration of young ovarian signaling. Though this method can provide insight into menopause associated increases in disease it can also be applied to other disease such as those associated with primary or secondary ovarian insufficiency.
Before beginning the procedure confirm the appropriate level of sedation by toe pinch. And apply ointment to the animal's eyes. Place the mouse in the supine position on a stack of heated paper towels and starting just below the ribs and moving distally use number 40 blade hair clippers to remove a two to three centimeter square of hair, a few millimeters lateral to the midline on each side of the animal with a one to two centimeter strip of hair covering the midline.
After administering analgesia transfer the animal onto a heated sterile surgical field in the supine position, and use a two by two centimeter piece of 70%ethanol soaked gauze followed by a Betadine to disinfect the surgical area. Next make a one to 1.5 centimeter paralumbar incision in one of the surgical areas and use scissors to blunt dissect the skin from the underlying fascia. Then make another incision through the fascia and blunt dissect laterally under the fascia as superficially as possible until the abdominal cavity is reached.
Place a two by two centimeter gauze surgical drape over the incision and wet the gauze with sterile saline. Using small forceps locate the adipose tissue that surrounds the ovary and gently extract the fat pad and ovary onto the drape. The ovarian fat pad will appear whiter than the surrounding adipose tissue.
Place a 1.5 inch bulldog clamp onto the fat pad taking care not to obstruct the ovarian bursa to hold the tract in place during the procedure. Use a dissecting microscope to identify the ovarian bursa. Then grasp the bursa with two watchmaker forceps and incise the bursa on the opposite side from the ovarian hilum to expose the ovary.
Before deciding where to open the bursa try to visualize a pocket in the bursa where you will place the new ovary prior to suturing. Only open the bursa as far as necessary to remove the endogenous ovary. Gently remove the ovary from the bursa and clamp the ovarian hilum with watchmaker forceps to excise the ovary and to prevent bleeding.
Then place the donor ovary in a watch glass containing four degrees celsius sterile saline and place a loose cover over the glass. After ovariectomizing the recipient mouse as just demonstrated place a small piece of hemostatic foam pad inside the empty bursa of the recipient animal to control bleeding and place a small piece of saline soaked gauze over the exposed surgical site. Load a castroviejo needle holder with a tapered needle threaded with a nine O or 10 O filament suture and glass bead for embedding.
Using a second pair of watchmaker forceps simultaneously remove the gauze and foam pad from the recipient bursa. Then pick the glass bead with a Bishop-Harmon forceps and drop the bead onto the fat pad next to the bursa to allow the bead to be rolled into the bursa. When the bead is finally in place close the bursa with one to two Halsted sutures and lightly irrigate the site with sterile saline.
After removing the fat pad clamp return the ovary and fat pad to the abdomen. Stay sutures may need to be placed to position the ovary for the subsequence placement of sutures for closing the bursa. Then use or vicryl sutures to close the abdominal wall and nine millimeter wound clips to close the skin.
Finally the animal in a heated recovery cage shielded from light with monitoring until full recovery. Post-reproductive female mice that receive new ovaries live significantly longer than mice that undergo a sham surgery. No difference is detected between ovaries transplanted at the 11 or 17 months of age.
However the depletion of germ cells prior to transplantation doubles the extension of the lifespan. The mean lifespan of CDAG female control mice is 644 days. Mice that undergo sham surgery at 11 months lived to an average age of 728 days.
Acyclic intact mice live an average of 810 days when transplanted at 11 months. And 798 days when transplanted at 17 months. When the young ovaries are depleted of germ cells prior to transplantation the mean life span is extended to 880 days, twice the lifespan extension seen in mice that receive young germ cell containing ovaries.
VCD treatment depletes primodial and primary follicles. The remaining secondary and antral follicles will then cycle and not be replaced, depleting the ovary of all germ cells. For example in this representative experiment ovaries collected from mice analyze 37 days after the initiation of VCD treatment demonstrate significantly depleted primordial and primary ovarian follicles compared to ovaries in control animals.
After watching this video you should have a good understanding of how to manipulate ovarian function to facilitate separation of reproductive aging from chronological aging and be able to identify the separate influence of each on longevity and health span.
