All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Establishment of Resiniferatoxin (RTX) Neuropathy
CAUTION: RTX is neurotoxic and hazardous. On contact, it acts as an irritant to the eyes, mucous membranes, and upper respiratory tract. Avoid inhalation and wear lab eyeglasses and coats during RTX preparation. Rinse with plenty of water in case of skin contact or after handling.
- Add 1 mg of RTX powder to a 200 µL mixture of equal volume of Tween 80 and absolute ethanol (100 µL for each solvent).
- Aliquot the RTX solution (12 µL/vial) and store it at -20 °C for up to 3 months. This constitutes the RTX stock; discard the remaining RTX solution at expiration.
- Dilute the RTX stock with normal saline to a final volume of 600 µL. The final concentration of the RTX solution should be 0.01%, which equals 1 µg RTX in 10 µL vehicle solution.
- Use 8-week-old adult male ICR mice (35-40 g) as experimental animals and administer a single dose of RTX solution (dose: 200, 50, and 10 µg/kg, respectively) intraperitoneally (i.p.) with a microinjection syringe to the mice. Mice were anesthetized by inhaler with 5 % isoflurane for deep anesthesia. If mice showed withdrawal action of extremities during RTX injection, mice should take longer inhalation of anesthesia.
Example: If the mouse weighs 40 g, then it will receive 20 µL of the RTX solution, representing the dose of 50 µg/kg.
- Give one group of mice an equal volume of vehicle (10% Tween 80 and 10% absolute ethanol in saline solution) as a control.
- After RTX injection, return the mice to a plastic cage on a 12-h light/12-h dark cycle and provide food and water ad libitum.
