Subcutaneous Dihydrotestosterone (DHT) Treatment: Mouse Model of Polycystic Ovary Syndrome (PCOS)

研究方案

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Create PCOS-like Mouse Model

NOTE: The mice used in this study were a mixed background (C57/B6, CD1, 129Sv) and were maintained with food and water ad libitum in a 14/10 h light/dark cycle at 24 °C.

1. DHT Pellet Preparation
   1. Autoclave silastic tubing to sterilize. Cut silastic tubes to a length of 15 mm with a razor blade.
   2. Seal one side by injecting medical adhesive silicone into the tube with a 20 G blunted needle attached to a 3 mL syringe. Plunger should be withdrawn from the syringe and then adhesive inserted into the reservoir. The plunger is reinserted and adhesive pushed down until it begins to emerge from the needle. The blunted needle is made by cutting the sharp tip side of 20 G needle with any strong scissors. The length of silicone in the tube should be more than 2 mm to allow for post-production trimming.
   3. Dry overnight; check for air bubbles on the sealed side. Those with no air bubbles are used for DHT pellets. Others can be used for no-DHT control pellets.
   4. Wear gloves, mask, goggles and lab coat before making DHT-pellet in a hood to avoid DHT exposure to skin.
   5. Pour DHT powder into a plastic weigh boat and press the open side of the adhesive capped tubes (produced in 1.1.1) into the DHT powder.
   6. DHT powder can be tamped down with a large paper clip that is straightened. Continue until DHT reaches a height of 4 mm (or desired length). Check the length of DHT with a ruler, and seal the open side with silicone, dry overnight.
   7. Cut each sealed side to make the length of silicone 2 mm long. The total length of pellet will be 8 mm.
   8. Seal both side of empty silastic tubing as control (no-DHT) pellet.
   9. Keep pellets in a 50 mL conical tube wrapped with foil (to prevent light exposure) at room temperature until use. The DHT pellets maintain full efficacy for at least 3 months of storage.
2. DHT Insertion and Replacement
   1. Up to 20 DHT pellets or control pellets are submerged separately in a 50 mL conical tube with 30 mL sterile 0.9% saline for 24 h at 37 °C for equilibration just before insertion.
   2. Prior to surgery, the surfaces and gloves are disinfected with clidox and the work surfaces are covered with clean plastic-backed absorbent paper (surgical pad). All instruments that come in contact with animals are decontaminated prior to entering the animal facility by autoclaving.  Instruments will be allowed to dry and cool prior to use.
   3. 2-month-old female mice are used (4–5 mice/cage). Mice are injected intraperitoneally with Xylazine (3.5 mg/kg bw) and Ketamine (78.8 mg/kg bw) using an insulin syringe. Calculations for mixing and dosing anesthesia are in Table 1. 
   4. After adequate anesthesia is achieved, as measured by loss of toe reflex and slowed breathing, the mouse will be prepped for surgery.
   5. Disinfect the skin of the area with betadine using sterile gauze and clean with 70% ethanol.  The area will again be painted with betadine.
   6. Cut a hole around 5 mm length with scissors under the skin near the neck. Using a 10 G trochar, make a small tunnel (15 mm) in the rostral direction. The pellet is inserted dorsally with the trochar.
   7. The opening is then sealed with surgical adhesive. Manually approximate the wound edges with forceps and gentle brushing strokes to apply a thin film of liquid adhesive to the approximated wound edges. Buildup 3 thin layers of adhesive to ensure the adhesive is evenly distributed over the wound. The adhesive should extend 1 cm on each side of the apposed wound edges. Suture or surgical clips can also be used to close the hole. Put mice back into cage, individually housed, with a heat pad for recovery. Replace pellet every 4 weeks to maintain a constant level of androgen exposure. The original pellet will be removed and new pellet will be inserted as described above, in a similar position.

Ketamine (100 mg/mL)	Xylazine (100 mg/mL)	Saline (0.9%)
175 μL	15 μL	810 μL
Intraperitoneal injection of working solution: 0.1 mL/20g body weight

Table 1. Ketamine/xylazine cocktail.

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材料

Name	Company	Catalog Number	Comments
Crystalline 5α-DHT powder	Sigma-Aldrich	A8380-1G
Dow Corning Silastic tubing	Fisher Scientific	11-189-15D	0.04in/1mm inner diameter x 0.085in/2.15mm outer diameter
Medical adhesive silicone	Factor II, InC.	A-100
Goggles, lab coats, gloves and masks
3 mL Syring	Becton, Dickinson and Company (BD)	30985
Attached needle: 20G	BD	305176
Ruler: any length than 10 cm with milimeter scale.
Xylazine	Vet one AnnSeA LA, MWI, Boise	NDC13985-704-10	100 mg/mL
Ketamine Hydrochloride	Hospira, Inc	NDC 0409-2051-05	100 mg/mL
Surgical staple	AutoClip® System, Fine Science Tool	12020-00
Insulin syringe	BD	329461	1/2 CC, low dose U-100 insulin syringe
Trocar	Innovative Research of America	MP-182
Razor blade	Fisher Scientific	12-640
Clidox	Fisher Scientific	NC0089321
Surgical underpad	Fisher Scientific	50587953	Andwin Scientific 56616018
Betadine Antiseptic Solution	Walgreens
3M Vetbond (n-butyl cyanoacrylate)	3M Science. Applied to Life