Development of an Uncomplicated Mild Traumatic Brain Injury Model Modified by Weight-Drop Method and Evidenced by Magnetic Resonance Imaging

Our research focuses on developing a closed-head injury animal model that mimics neuroimaging outcomes of uncomplicated mild traumatic brain injury. We end to determine whether different impact parameters in RmTBI lead to distinct imaging, behavioral, and pathological changes. We developed an animal model that replicates the radiological changes of uncomplicated mTBI, demonstrating significant behavioral deficits and long-term brain atrophy.

This work connects cross-sectional human studies with animal pathology research, providing translation and longitudinal neuroimaging assessment to better understand the mTBI progression. Our results open new avenues for investigating disease progression and outcome changes after uncomplicated mTBI. They also highlight the crucial role of injury parameters in shaping post-injury outcomes.

Our lab focuses on pediatric and adolescent mTBI, the largest patient cohort. We'll apply our comfortable CHI model to study developmental brain injury and track long-term changes using MRI, behavioral, and neuropathological assessments. To begin, place the anesthetized rat on a heating pad and secure it in the stereotaxic frame using a tooth bar.

Position the ear bars to stabilize the head. Ensure the rat is centered and symmetrical in the stereotaxic frame. Now, attach the sensor of the pulse oximeter to the hind paw to monitor respiration rate, heart rate, blood oxygen level, and body temperature.

Apply hair removal cream to the head After three minutes, wipe off the cream using 70%isopropyl alcohol swabs. Clean the shaved area multiple times using a sterile cotton swab soaked in iodine. Then, remove the iodine residue with a cotton swab soaked in 70%ethanol.

Using sterile surgical blade, create a midline incision approximately two to 2.5 centimeters in length to access the skull surface. Next, remove the soft tissue from the skull using a cotton pad. Clean the skull surface with a cotton swab soaked in 0.9%saline, followed by a dry cotton pad.

Now, identify the bregma as the reference point to determine the impact area. After identifying the desired coordinates, cement a circular stainless steel helmet over the designated area using dental cement. Then, remove the heating pad and pulse oximeter.

Move the stereotactic device and the rat onto the lift table under the closed head injury impactor. Elevate the rat's body using a foam sponge. Remove the rat from the ear bars while keeping it secured on the tooth bar connected to a nose cone delivering 2%isofluorine.

Ensure the rat's head and body are level in the rostrocaudal direction. Adjust the lift table to ensure there is no gap between the CHI impactor and the helmet. Then, turn off isoflurane immediately before the impact.

Now, drop a 600 gram brass weight from a height of one meter through a stainless steel tube to the secured impactor with a round tip, aiming at the metal helmet. After striking, lower the lift table. Remove the rat from the stereotaxic frame and place it in a supine position on a heating pad.

Record the righting reflex time, which is the time when the rat attempts to move from the supine position to the prone position. Now, remobilize the rat in the stereotaxic frame. Remove the helmet and clean all the connective tissue before cementing from the skull surface.

Now, cover the skull with dental cement and allow it to dry. Using the back of a tweezer, check that the cement is stiff and hard. Then, close the incision using 4o nylon surgical sutures with four to five independent knots and apply topical antibiotics to the surgical site to prevent infections.

Inject one milliliter per kilogram body weight of carprofen subcutaneously into the neck as a post-surgery analgesic. Place the rat in a clean cage on a heating pad until it regains consciousness. Once the rat sits upright, return it to the home cage.

Orally administer five milliliters of acetaminophen mixed in 200 milliliters of water daily for three consecutive days as an analgesic. Position the anesthetized rat in the head holder and connect to a nose cone for maintenance of anesthesia. Fixate the head with a small piece of tape to prevent movement during scanning.

Then, place a pressure pad under the thorax to monitor respiration. Insert the electrodes and tape the oximeter clips to the hind limb to check the heart rate. Insert the rectal probe to measure the rectal temperature.

Cover the rat with a heating blanket with circulating warm water and tissue wrap to maintain body temperature during the experiment. Use the laser positioning system of the PET MR scanner to mark the center of the head for precise alignment. Move the rat into the MRI bore using the motorized animal transport system until the center of the head aligns with the isocenter of the scanner.

Finally, obtain the MRI images and analyze them. No significant skull fracture, brain contusion, white matter edema, or deformation was observed in T2 weighted images and fractional anisotropy maps at one and 50 days post-CHI, confirming minimal structural damage in the CHI model. A significant reduction in cortical volume was observed at 50 days post-CHI, and repetitive CHI resulted in greater cortical loss compared to single CHI.

The most substantial cortical volume reduction was noted in slices at bregma minus four to plus zero and bregma minus five to plus one after repetitive CHI with different impact parameters. A significantly smaller cortical volume was observed at bregma zero after central brain CHI compared to SMCx CHI. Immunostaining at day 50 post-injury demonstrated astrocyte accumulation in the ipsilesional SMCx, irrespective of CHI severity and impact site.