Generating a Glioblastoma Rat Model

Protokół

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

1. F98 GB Rat Cell Model

1. Culture the F98 GB cells, obtained from ATCC, in monolayers using Dulbecco's modified Eagle Medium, 10% calf serum, 1% penicillin, 1% streptomycin, 1% L-glutamine, and 0.1% amphotericin b, and place in a CO₂ incubator (5% CO₂ and 37 °C).
2. Inoculate the glioma cells in the brain of female Fischer F344 rats (body weight 170 g).
   1. Use sterile instruments and wear sterile gloves at all times.
   2. Anesthetize the rats by injecting a mixture of 74 mg/kg ketamine and 11 mg/kg xylazine intraperitoneally (IP) with an insulin syringe (1 mL, 29 G). Confirm the anesthetization by the absence of response to the withdrawal reflex of the limb. Immobilize the rats in a stereotactic device using fixation points for the nose and ears. Place a carbomer eye gel to prevent dryness of the eyes while under anesthesia.
   3. Shave the rat from eye level to the back of the skull and disinfect the skin with povidone-iodine.
   4. Expose the skull through a midline scalp incision of 2 cm, and make a 1 mm hole (diamond drill) 2 mm posterior and 2.5 mm lateral to the bregma in the right frontal hemisphere.
   5. Insert a stereotactically guided insulin needle (29 G) and inject 5 µL cell suspension (20,000 F98 GB cells) 3 mm deep using a micro-syringe pump controller (settings: inject (I50), rate 1 nL/s (001 SDN)).
   6. Withdraw the syringe slowly and close the incision with bone wax. Suture the skin and disinfect it with povidone-iodine.
   7. Stabilize the body temperature of the animal post-surgery using a red lamp. Monitor the awakening of the rat until it has regained sufficient consciousness to maintain sternal recumbency. Do not return the animal to the company of other animals until fully recovered. Keep all animals under environmentally controlled conditions (12 h normal light/dark cycles, 20-24 °C, and 40-70% relative humidity) with food and water ad libitum. Make sure to follow the animals closely by monitoring their body weight, food, water intake, and their activity and normal behavior. Use a lethal dose of pentobarbital sodium to euthanize the animals (160 mg/kg) if a decline of 20% in body weight is observed or when the normal behavior severely deteriorates (e.g., lack of grooming).

Materiały