To begin, randomly allocate the mice into either the control or repetitive mild traumatic brain injury group. Acclimate the mice in separate cages one week before the experiment. Carefully inspect the modified fluid percussion device for signs of leakage and ensure that there are no bubbles present within the system.
After anesthetizing and trimming the fur on the mouse's head, disinfect the scalp with 75%ethanol. Secure the mouse using non-terminal ear bars in a stereotaxic frame and place an isothermal heating pad underneath to maintain body temperature at 37 degrees Celsius. Carefully clean the surgical area using a saline-soaked cotton swab.
After making a 1.5-centimeter incision along the midline of the scalp, use a flat-tipped micro drill bit and a microsurgical blade to create a thin skull window in the right frontal motor cortex. Locate the surgical site at 1.5 millimeters anterior to the bregma and 1.3 to 2.0 millimeters lateral to the midline. Attach an adjusted female Luer lock to the thin skull site.
Transfer the mouse from the stereotaxic apparatus to the Impactor platform. Connect the female Luer lock to the male Luer lock at the end of the fluid percussion injury device tubing. To apply fluid percussion injury impact, raise the pendulum to the designated degree along the protractor on the device and release the pendulum using software control, achieving a percussion intensity of approximately two atmospheres.
Following the impact, immediately detach the Luer lock connection and transfer the mouse to an isothermal heating pad for recovery. After the second traumatic brain injury, carefully remove the female Luer lock and dental cement. Suture the scalp with tissue adhesive and use flat forceps to pinch the scalp to facilitate the adhesive process.
To alleviate post-surgical pain and discomfort, apply a one-to-one mixture of erythromycin and sodium diclofenac ointment to the wound. Finally, transfer the mouse to an isothermal heating pad for recovery. Analysis at seven to 12 days post-injury using the Morris water maze showed that repeated mild traumatic brain injury led to impairments in spatial learning and memory in mice.
The open field test conducted at six days post-injury showed no significant impact on the mice's general locomotive abilities, but induced significant anxiety and altered exploratory behavior in repeated mild traumatic brain injury mice. The Y-maze test performed at eight days post-injury revealed impairments in both spatial working and reference memory in repeated mild traumatic brain injury mice.
