Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury

Our protocol is the first to detail a mouse model of post-traumatic epilepsy induced by mild traumatic brain injury. The use of a mild diffused traumatic brain injury model mimics an important aspect of the pathology of human traumatic brain injury which almost always has a diffused component. In order to develop biomarkers and treatments, research using models that appropriately reproduce aspects of human pathology is needed.

Our traumatic brain injury model also has the potential to provide insight into the mechanisms that cause impairment of brain function after mild traumatic brain injury or concussion. After confirming a lack of response to pain reflex, place the mouse onto a foam pad and position the head of the mouse under the weight drop tube. Place a flat 1.3 centimeter diameter one millimeter thick 880 milligram weight stainless steel disc in the center of the animal's head between the eyes and the ears.

Remove the pin in the weight drop tube to release the 100 gram weight rod from a height of 50 centimeters. To induce a sham injury in the control mice, remove the weight rod from the tube to prevent accidental release of the pin during weight drop. After weight drop induction, place the animal on its back on a heating pad covered with a sterile poly lined absorbent towel with monitoring until full recumbency.

Then place the mouse in a clean cage that has been warmed on a heating pad with recovery gel and a few moistened chow pieces for 45 minutes. To prepare the surgical field for EEG electrode implantation, place a temperature sensor onto the pad and place the re-anesthetized brain injured mouse onto a sterile drape on top of the heating pad on the stereotactic apparatus. After fixing the head in place with ear bars, apply lubricating ointment to the animal's eyes and use depilatory cream to remove the hair from the scalp.

Disinfect the exposed skin with three 20-second alternating swabs of povidone iodine surgical antiseptic solution and 70%ethanol in a circular motion. After removing the scalp, apply small hemostats on the opened skin borders to expand the exposed area. Next, use the scalpel to gently remove the periosteum and swab the cranium with hydrogen peroxide.

When the cranium is cleared of tissue and exhibits a whitish appearance, dry the bone with a sterile gauze or swab. For electrode implantation, use a high-speed drill with a round 0.5 millimeter steel bit at 5, 000 to 6, 000 rotations per minute to create six burr holes for the screw electrodes. When all of the holes have been drilled, place the six screw electrodes into the burr holes and mix a half scoop of dental cement powder with several drops of solvent.

Use a spatula to stir until the dental cement mixture is tacky but malleable and stiff enough to be properly condensed when placed on the animal's cranium. Apply the cement over the entire exposed surface of the cranium and each screw electrode. After one to two minutes, turn on the soldering iron and place the three EEG head mount into a stereotactic holder arm.

Position the head mount over the skull so that the six wire lead positions match the position of the wire lead of each screw electrode. Lower the device so that its ventral part rests on top of the dental cement. Twist the wire of each lead from each of the screw electrodes with the corresponding wire lead of the head mount, solder each twisted pair of wire for proper signal conduction and use scissors to trim any excess wire.

Bend each soldered pair of wire leads around the head mount. When all of the pairs have been bent, cover all of the wire with fresh dental cement leaving only the black portion of the head mount exposed. Release the hemostats holding the skin flaps and apply chlorhexidine antiseptic to the area around the implant to avoid infection.

Then transfer the animal from the apparatus to a balance to measure the animal's weight as a reference for future monitoring before placing the animal in a clean cage on a warm heating pad with monitoring, recovery gel, and a few moistened chow pieces. In this figure, a spontaneous seizure in a mouse 97 days after repeated weight drop traumatic brain injury recorded using a three EEG head mount configured as demonstrated in this video protocol is shown. Here, a spontaneous non-convulsive electrographic seizure recorded in a mouse 65 days after repeated weight drop traumatic brain injury is shown.

In this figure, a spontaneous seizure in a mouse 23 days after repeated weight drop traumatic brain injury was recorded using a one EEG head mount. The spectral power of a typical seizure indicates the highest density to be in the range of 10 to 40 hertz with a peak at 15 hertz. This protocol demonstrates the timelines for seizure onset in animals after repetitive weight drop traumatic brain injury demonstrating the incidence of seizure clustering in some animals and emphasizing the importance of acquiring continuous rather than intermittent recordings.

Proper placement of the steel disc, careful implantation of the screw electrodes and careful soldering of the wire leads are all crucial for a successful long-term experiment. After EEG monitoring, animals with brain injury that develop seizures can be stratified from those that do not for subsequent analysis of their cellular, molecular, and physiological differences. To stratify animals with post-traumatic epilepsy from those that incur traumatic brain injury but do not develop seizures is critical for the development of interventions and biomarkers.