Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

In this study, we introduced a closed-head mild traumatic brain injury rat model, and provided a comprehensive overview of its validation. Our goal was to offer a straightforward approach for replicating this type of brain injury and a rapid assessment method for confirming the success of the model. This model successfully simulates a closed-head mild traumatic brain injury without the need both gap incision or score opening, and provide a more accurate representation of the impact scenario observed in human cases.

The avoidance of scab incision helps to prevent inflammatory responses that may not align with the actual situation. the traumatic brain injury lacks objective and accurate diagnostic methods in the future. Our lecture will continue to explore possible biomolecular markers and their deep molecular and help to transform the obtained molecular markers into clinical and forensic identification practices.

To begin, place a 12 centimeter thick sponge with a 35D hardness in an acrylic box lacking a top cover. Then cut a sheet of tinfoil and adhere it to the acrylic box with adhesive tape to create a fragile surface capable of supporting the weight of a rat. Also, mark a 10 centimeter long line as the designated location for positioning the rat's head.

Using an iron stand, firmly secure a polyvinyl chloride, or PVC, tube in place. Prepare a perforated weight weighing 550 grams with an 18 millimeter diameter. Attach the weight to a fishing line at a height of one meter inside the PVC tube and adjust the position of the guide tube three centimeters above the tin foil.

Next, make a helmet from a stainless steel disc. Prepare a wedge-shaped sponge pillow to place under the rat's head. Swiftly positioned the anesthetized rat on its chest in the tin foil.

Slide the pillow beneath the rat, ensuring its head remains parallel to the foil surface. Align the helmet with the rat's ears, and securely fasten it in place. Confirm that the PVC pipe is directly above the helmet.

Then release the weight and allow it to impact the rat's head resulting in a fall onto the sponge and a 180 degree rotation. Afterwards, position the rat on its back inside a clean cage. After inducing a mild traumatic brain injury in an anesthetized rat, position it on its back inside a clean cage.

Record the duration from when the rat is anesthetized to when it regains the writhing reflex. Also, record the time it takes for the rat to exhibit its first seeking behavior from the moment of anesthesia. Next, place the rat in the center of a circular apparatus with an exit.

Measure and record the time it takes for the rat to exit the circle. Then place the rat successively on three beams of varying length each for a duration of one minute. Score the rat on a scale of zero to six, depending on their performance on the beam.

mTBI rats exhibited a significantly prolonged recovery from unconsciousness. The seeking behavior increased significantly in the mTBI group during the recovery period. Also, the mTBI rats took longer to exit the circle compared to Sham rats.

The beam balance test showed a significant injury effect in wide beam tasks, indicating balance impairment in mTBI rats 10 minutes post impact. The 2.0 and 1.5 centimeter wide beams effectively differentiated between Sham and mTBI groups in three independent experiments. After inducing a mild traumatic brain injury in an anesthetized rat, position it on its back inside a clean cage.

Place foam padding beneath the balance beam to reduce the risk of injury to the rats in case of falls during the test. Place an escape box at one end of the beam. Activate the video camera to record the test.

Once the animal recovers, position the rats at the other end of the balance beam. Position the rats on the two centimeter wide beam for a series of five consecutive trials. Record the initiation and conclusion of each trial when the rat's nose crosses the starting and finishing lines respectively.

Prepare the open field arena ensuring it is clean and devoid of any prior odor cues. Divide the arena into three distinct zones:a central inner zone, a middle zone, and an outer zone. Place a rat in the center of the open field arena and initiate the timer.

Allow the rat to freely explore the arena for a duration of five minutes. After this period, carefully and gently return the rat to its home cage. Quantify the total distance covered by the rat during the five minute exploration period.

Additionally, determine the amount of time the rat spends in the three zones. Ensure that the water maze apparatus is in proper working condition. Place the platform at a depth of 2.5 centimeters below the water surface.

Dye the water black, and position cues in the four cardinal directions. Establish a monitoring system to record and observe the behavior of the rats. Then introduce the rat into the water maze.

If the rat is unable to reach the platform within a two minute timeframe, gently guide it using a wooden stick. Allow the rat to become accustomed to the maze environment while standing on the platform for a duration of 20 seconds. Then remove it.

On the sixth day, which is the probe test day, remove the platform from the water maze and position the rat in the same quadrant for a duration of two minutes. Employ the monitoring system to continuously observe and record the rat's behavior. Motor coordination was assessed pre-and post-anesthesia or injury using the beam task.

At day one, post-injury, mTBI rats showed significantly more hind-limb slips compared to Shams. However, after two days hind-limb slips returned to Sham levels. All six mTBI rats had more post impact hind-limb slips with no differences in beam traversal times between mTBI and Sham rats.

Distance traveled showed no significant differences between sham and mTBI groups. Anxiety-like behavior was evident in mTBI rats at three and seven days post-injury spending less time in the center zone during the open field test. Morris Water Maze results indicated impaired spatial learning and memory in mTBI rats.

They took longer to find the hidden platform and spent less time searching for the removed platform during the probe trial without differences in swimming speed suggesting no impact on spontaneous locomotor function.