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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal care
- Use 5- to 6-week-old male C57BL/6J mice (20-25 g of weight) in this experiment.
- Maintain mice on a 12 h/12 h light/dark cycle, and make sure they receive food and water ad libitum. Provide the same amounts of food and water to both the sham and TBI groups throughout the study.
- Make all efforts to minimize the animal pain and discomfort.
2. Induction of traumatic brain injury
- Inject Ketamine (80-100 mg/kg)/Xylazine (10 mg/kg) IP for anesthesia. Test the depth of anesthesia using an eye reflex or pain reflex. Use artificial tears or lubricant eye ointment to keep the eyes from drying.
- After anesthesia, put the mouse in a prone position. Use a temperature-controlled heating pad to maintain the temperature at 37 °C during the surgery and for 30 min after TBI.
- Shave the hair of the incision area.
- Disinfect the scalp with 70% ethanol using three alternating scrubs, then incise the scalp in a sagittal plane.
- Use forceps to retract the incision on both sides and separate the periosteum slightly.
- Use a marker to draw a circle (3 mm diameter) on the right parietal area of the skull, 2 mm away from the midline.
- Drill the skull with an electric drill. Ensure that this step is operated carefully to protect the dura from being damaged.
- Remove the bone flap and expose a small bone window (3 mm in diameter).
- Place a plastic injury cannula (internal diameter = 2.5 mm, length = 8 mm) over the craniotomy and cement the cannula to the skull using a dental acrylic.
- Fill the cannula with sterile 0.9% NaCl (normal saline) using a syringe (5 mL) to ensure that there are no bubbles in the cannula.
- Turn on the oscilloscope and amplifier and ensure that the high-pressure tube of the LFPI device is free of air bubbles. Test the device by delivering about 10 pulses until it gives a steady signal. Adjust the angle of the pendulum starting position to reach a pulse intensity of about 2.0 atm.
- Connect the injury cannula to the LFPI device. Induce brain injury by pulling the trigger and releasing the pendulum. Then, obtain a pulse and transmit it to the dura through the entire closed fluid-filled tubing system.
- Operate the mice in the sham group with the same surgical procedure. Do not perform the LFPI.