This protocol provides an opportunity to harmonize studies which use the rat lateral fluid percussion injury model of traumatic brain injury in combination with EEG recording via wireless telemetry. It can used to investigate factors influencing post-traumatic epileptogenesis and to test the neurotherapeutic potential of drug interventions, which may prevent the development of post-traumatic epilepsy. This approach permits the long-tern video EEG recording of freely moving rats and allows for the moderate manipulation of animals without the interruption of EEG recording.
Demonstrating the procedure will be Matthew McGuire, an MD-PhD student from my laboratory. See the manuscript accompanying this protocol for detailed surgical procedures. Make a 1 1/2 to 2 1/2-centimeter midline incision through the skin and muscle of the scalp using a number 10 scalpel blade.
Retract the skin and muscle to expose the skull and provide a clear surgical field. Electrocautery is useful for achieving quick hemostasis. Next, shave down the lateral ridge of the left parietal bone using a surgical curette to produce a smooth, flat surface so that the base of the female-female Luer lock hub can rest flush with the skull.
Irrigate the skull surface and surrounding tissues with 2.0 milligrams per milliliter gentamicin solution in sterile saline, and blot excess solution with sterile swabs. Then, apply 3%hydrogen peroxide to the skull to dry the bone. At this point, create a five-millimeter diameter craniectomy site through the left parietal bone.
Then, remove the bone flap with the surgical curette and smooth tissue forceps. Next, using a stereo microscope, gently remove the thin rim of bone remaining with smooth tissue forceps, taking care not to rupture the dura. Then, swab the skull with 70%ethanol to remove any bone dust and to dry the skull.
Apply a thin layer of cyanoacrylate glue around the bottom edge of the Luer lock hub, and secure it to the skull over the craniectomy without obstructing the opening and without allowing the glue to contact the dura. Then, seal the Luer lock in place with a thin layer of glue around the outside base of the hub. Next, prepare a slurry of dental cement, and apply this to the surface of the skull around and over the base of the Luer lock hub to secure it in place.
Then, fill the Luer lock hub with a sterile preservative-free solution containing multiple electrolytes, using a syringe and needle. A convex bolus of saline should be seen above the top of the rim. Once the dental cement is completely cured, discontinue gas anesthesia and remove the rat from the stereotaxic frame.
Place the rat on a platform next to the fluid percussion injury device in sternal recumbency. Then, secure a 12-centimeter length of pressure tubing to the end of the device's curved tip, with the opposite end terminating in a two-centimeter, male Luer lock twist connector. Secure the rat to the device by connecting the female end of the hub on the rat's skull to the male connector.
Repeatedly check the animal for return of withdrawal reflex. As soon as the rat regains withdrawal reflex but is still sedated, release the pendulum of the device to cause a single 20-millisecond pressure pulse and induce injury. Then, immediately disconnect the rat from the FPI device, place it in sternal recumbency, and provide supplemental oxygen via a nose cone until spontaneous breathing returns.
Note that apnea is an anticipated consequence of the injury. If necessary, provide periodic manual breaths via a bag valve mask until the rat begins to spontaneously breathe on its own. Monitor continuously, and record the time of return of righting reflex.
Four hours after injury, again anesthetize the rat, and place it back into the stereotactic frame to remove the Luer lock hub and dental cement. Apply a small drop of 0.5%bupivacaine hydrochloride to the skull in each of the locations where five pilot holes are to be drilled. Then, drill the pilot holes through the skull with a handheld 0.1-millimeter drill bit.
Next, secure the stainless-steel electrode screws. First, place a reference screw caudal to the lambda, over the cerebellum. Then, place recording electrodes in the four locations as seen here.
Be sure to swab the skull with 70%ethanol to remove any bone dust. Then, cover the craniectomy site with a thin layer of sterile bone wax to cover the exposed dura. Now, connect an electrode array to the five EEG electrodes by wrapping the exposed end of a color-coded electrode wire tightly around its designated stainless-steel electrode screw.
Collect the electrode wires into a coil underneath the pedestal, and secure the wires and pedestal into place with bone cement. Hold the pedestal in position until the bone cement has cured. Finally, attach the wireless transmitter with fresh batteries to the pedestal before removing the animal from the stereotactic frame.
Beginning on the day of injury, use the EEG manufacturer's collection software to continuously record video EEG, linking each wireless transmitter via a unique frequency to a specific receiver. Record video of each rat with its own camera configured to record at 30 frames per second. Manually screen through the EEG recordings to identify index events that define seizure activity.
This figure shows unilateral, intermittent delta slowing collected on the day of a moderate TBI. Here, we see a 90-second EEG trace from a sham-operated, uninjured control rat with FFT analysis of 2, 048-millisecond selected EPOC. Then, here we see an EEG trace of a moderately injured animal, which demonstrates the intermittent, unilateral delta slowing pattern and FFT analysis of 2, 048-millisecond selected EPOC.
This figure shows bilateral, continuous delta slowing collected on the day of a severe TBI, using the same analysis techniques. Here, we see a 90-second EEG trace, which demonstrates the continuous, bilateral delta slowing pattern of a severely injured animal. Here, we see nonconvulsive electrographic seizure activity collected three days post-severe TBI.
Data from a control rat three days after surgery is shown, as well as a 90-second EEG trace three days post-severe injury, and FFT analysis of a 2, 048-millisecond selected EPOC. And finally, this figure shows convulsive electrographic seizure activity collected nine days post-TBI, with FFT analysis from this animal. Here, we also see representative images of occasional intermittent signal dropout and loss of signal due to battery failure.
It is important to ensure that the dura is not disrupted and remains intact after the craniectomy and that the Luer lock hub is securely sealed to the skull. It is also important to make sure that the electrode wires make good contact with the recording screws that are placed in the skull. Finally, make sure the skull is free of dust and dry to ensure that the bone cement adheres to the head long term.
