A Pediatric Concussion Model in Mice: Closed Head Injury with Long-Term Disorders (CHILD)

During the last decade, models of mild traumatic brain injury have been developed in rodents with closed head injury. However, implementation of these models in developing rodents is sparse compared to the adult TBI literature. To address with clinical relevance, this study developed a closed head injury with long-term disorders, or CHILD TBI model.

For a long time, traumatic brain injury has focused on the consequences of severe brain injuries. In the last two decades, mild TBI effects have been reported, but few studies have assessed the long-term consequences. With a new development in neuroimaging, there's increased interest in the long-term outcomes which have been missing for pediatric models.

CHILD was designed to fill this gap. Using the CHILD model, we have characterized progressive and persistent changes within the brain such as neuronal deaths, altered diffusion MRI, modified neuronal activity and plasticity, increased gliosis, progressive behavioral perturbations, and cardiac dysfunction with age. Our new CHILD model presents the advantage of being performed and reproducible in different labs.

With wide adoption, we could expect an extension in knowledge about the long-term consequences of pediatric concussion. Using the CHILD model, we are continuing to investigate the effects and consequences of repetitive stress. We have already started to pursue how the combined effects of early infection after birth and CHILD may contribute to long-term outcomes.

Additionally, we observe protection of the vascular bed after repeated stress, suggesting that preconditioning after early infection may be occurring, but this await further investigation. To begin, warm the chamber for anesthesia induction and the recovery cages in advance using warming pads to prevent hypothermia. Set the stereotaxic apparatus with the impactor mounted at a 90 degree angle.

Verify that all parts of the stereotaxic apparatus are properly tightened and secured. Confirm that the piston is firmly screwed into the impactor. Next, cut an aluminum sheet and wrap the stereotaxic frame with foil to create a pad for the animal to rest on.

Secure the position and tension of the foil using laboratory tape to support the animal's weight. Afterward, turn on the impactor controller and adjust the impact speed to two meters per second for grade one or three meters per second for grade two, based on the severity of the injury. Then, weigh the animals on a weighing balance.

After anesthetizing the mouse, position the head of the mouse under the impactor. Ensure the pup is fully stretched out in length. Confirm that the three millimeter diameter impactor tip is covered with rubber to minimize metal contact with the skull.

Now, switch the knob to the Extend position to lower the impactor. Roughly lower the impactor, so that it is positioned between the ears of the mouse. Then, slide the mouse so that the piston tip is moved forward the equivalent of one piston tip length and one tip length to the left of the animal.

Set the impactor to the Retract position. Then, adjust the piston position to determine the severity of the injury. Set the dwell time to 0.1 seconds, regardless of the injury severity.

Press Impact on the right knob to initiate the impact. Rapidly, turn the left knob to the Off position to prevent system overheating. Then, place the mouse on its right flank in a recovery cage.

Monitor and record the writing time when the mouse rears on all four legs. Note the time it resumes exploratory behavior. Once fully recovered, return the mouse to its home cage.

Wash the brain sections four times for 10 minutes each in PBS under agitation. Transfer the brain sections into the blocking solution and incubate for 10 minutes at room temperature. Take the diluted primary antibodies in the blocking solution and add the brain sections.

Incubate the sections overnight at four degrees Celsius. After incubation, wash the sections four times for 10 minutes each in PBS under agitation. Transfer the sections into blocking solution containing secondary fluorescent antibodies diluted 1 to 1000, and incubate for one hour at room temperature.

After washing the sections four times in PBS, mount them onto slides using a mounting medium. Add a cover slip to each slide before storing them at four degrees Celsius until image acquisition. Acquire immunofluorescence images using an epifluorescence microscope and Micro-Manager software.

Female mice in the G2 group exhibited significantly longer time to stand than males within the same group, and both sexes showed prolonged times compared to sham controls, with greater differences between G1 and G2 severity levels. Female mice in the G2 group exhibited significantly longer time to explore compared to males and both sexes had higher times compared to sham controls, with significant severity-dependent increases between G1 and G2.Astrogliosis, evidenced by increased glial fibrillary acidic protein staining, was observed in the ipsilateral somatosensory cortex of the G2 group compared to sham controls at one day post-injury. NeuN immunohistochemistry showed no significant neuronal loss at one day post-injury.