Subcutaneous Dihydrotestosterone (DHT) Treatment: Mouse Model of Polycystic Ovary Syndrome (PCOS)
Transcript
For dihydrotestosterone or DHT pellet preparation, use a razor blade to cut a 15-millimeter piece of silicone tubing and use a 3-milliliter syringe equipped to a blunted 20-gauge needle to inject 2 to 5 millimeters medical adhesive silicone into one end of the tube. Allow the tubing to dry overnight and check for air bubbles on the sealed end.
Wearing the appropriate personal protective equipment, pour DHT powder into a plastic weigh boat in a flow hood. Impress the open end of the adhesive-capped tube into the powder. Using a large straightened paper clip, tamp the powder down into the tubing until the DHT reaches the appropriate experimental depth. Then, seal the open side of the tube with silicone overnight.
The next morning, cut each sealed side to obtain a 2-millimeter length of silicone on both sides of the DHT and no DHT control pellets. Then, store the pellets in a 50-milliliter conical tube wrapped with foil at room temperature for up to 3 months.
For PCOS mouse model generation, equilibrate up to 20 DHT or control pellets in separate 50-milliliter conical tubes containing 30 milliliters of 0.9% saline for 24 hours at 37 degrees Celsius. The next day, confirm a lack of response to toe pinch in an anesthetized 2-month-old female mouse and use sterile gauze to administer sequential povidone-iodine and 70% ethanol scrubs to the surgical area.
Next, make a 5-millimeter incision through the skin near the neck and use a 10-gauge trochar to make a 15-millimeter tunnel in the rostral direction. Use the trochar to insert a pellet dorsally into the tunnel and seal the incision with the surgical adhesive. Manually approximate the wound edges with forceps and use gentle brushing strokes to apply 3 thin film layers of liquid adhesive to 1 centimeter out from each side of the apposed wound edges.
Then, place the mouse alone in a cage on a heat pad with monitoring until full recumbency, replacing the pellet every 4 weeks to maintain a constant level of androgen exposure.
