Traumatic brain injury (TBI) is a critical public health issue worldwide and a leading cause of death and disability across all age groups [1]. The World Health Organization has predicted that TBI will be a major cause of death and disability by 2030, particularly in children and young adults, due to falls, road traffic accidents, and sports injuries [2]. Pediatric TBI is particularly concerning because the brains of children are undergoing rapid growth and forming complex neural networks [3,4]. This period of neurodevelopment is essential for cognitive, behavioral, and motor functions, and any disruption during this time could have significant long-term consequences [5].

The developing brain is particularly vulnerable to injury due to its plasticity and ongoing myelination, synaptogenesis, and pruning processes [6–8]. The same traumatic event can lead to different outcomes depending on the age at which the injury occurs. Pediatric brains are more susceptible to injury mechanisms such as diffuse axonal injury, swelling, and metabolic crisis, which can result in more severe primary and secondary damage compared with adults [9–13]. Despite these differences, little research has been conducted on the age-dependent outcomes of TBI, particularly in terms of secondary injury mechanisms such as neuroinflammation and apoptosis. This study seeks to fill this gap by comparing outcomes in pediatric and adult mice subjected to moderate to severe TBI

TBI outcomes are influenced by complex, multifactorial processes, including the heterogeneous nature of the human population, different injury types and severity, and the timing and characteristics of post-injury clinical care [14]. Although age-related differences in pathophysiological processes after TBI are not well understood, TBI involves a complex pathophysiological process with several overlapping phases, including primary injury, secondary injury, and regeneration. Following a primary injury, such as focal hematoma, contusion, or diffuse injury, secondary injury mechanisms are initiated over hours to days through complex biochemical and physiological processes. This secondary injury could be exacerbated by aging [15,16].

TBI results in temporary or permanent physical, cognitive, behavioral, and emotional impairments [17]. The underlying pathophysiology of neurobehavioral changes after TBI involves numerous biochemical processes initiated by the trauma. The processes leading to secondary brain damage include the release of neurotoxic endproducts, activation of neuroinflammation, formation of reactive oxygen species, and neuronal cell apoptosis [18–20]. Neuroinflammation is a component of both acute and chronic injuries following TBI [21]. The initiation of inflammatory responses involves the activation of glia, both astrocytes and microglia, which release various factors that recruit the inflammatory response [22,23]. Apoptosis, or programmed cell death, also contributes to neuronal loss and functional deficits. Heat shock proteins, such as heat shock protein 70 (HSP70), play a significant role in cellular protection by assisting in protein folding, preventing aggregation, and modulating stress responses [24]. We previously reported that neurobehavioral deficits following TBI may result partly from increased neuroinflammation in the damaged cerebral cortex [25]. Age-dependent differences in the rate of loss of cortical mantle volume during the acute stage following TBI have also been demonstrated [26].

Despite the growing body of research on TBI, most studies have focused on adult models or failed to account for age at injury differences. Pediatric brains, with their unique structural and functional characteristics, require special attention to determine how secondary injury processes such as neuroinflammation and apoptosis differ from those in adults. Understanding these differences is critical to developing age-specific therapeutic interventions that target neuroinflammatory pathways and apoptotic processes, and to minimizing long-term cognitive and behavioral impairments. By comparing neuroinflammatory, apoptotic, and stress responses between pediatric and adult mice following TBI, this study contributes crucial knowledge that can guide the application of future therapeutic strategies for pediatric neurotrauma. We investigated time-dependent neuroinflammatory responses, neuroglial apoptosis, and stress responses in pediatric and adult male mice exposed to moderate to severe controlled cortical impact (CCI). By elucidating the age-dependent differences, this study emphasizes the need for age-specific therapeutic interventions that can mitigate long-term damage and enhance recovery.

The present study investigated the time-dependent effects of TBI on neuroinflammation, apoptosis, and stress response in pediatric and adult mice. Our findings reveal that pediatric and adult mice showed significant time-dependent differences in the severity of these responses with a similar pattern. However, pediatric mice exhibited more early intense inflammatory response and apoptotic activities compared to adult mice. Additionally, both age groups showed a significant but transient increase in HSP70 expression, suggesting an acute stress response post-injury.

The present study provides important insights into the time-dependent effects of TBI on neuroinflammation, apoptosis, and stress responses in pediatric and adult mice. Our findings revealed that pediatric mice exhibit a more early pronounced neuroinflammatory and apoptotic response compared with adult mice, indicating the vulnerability of the developing brain to TBI. Additionally, both age groups demonstrated a transient increase in HSP70 expression, indicating a robust acute stress response. These results emphasize the need for time- and age-specific therapeutic strategies to mitigate the long-term consequences of TBI and enhance recovery outcomes.

Neuroinflammation is a prominent short- and long-term consequence of neuronal injuries that occur after TBI. It involves the activation of glia, including microglia and astrocytes, which release inflammatory mediators within the brain, and the subsequent recruitment of peripheral immune cells [21]. Various animal models have helped elucidate the pathophysiology of TBI and assess the safety and efficacy of novel therapies prior to clinical trials [21]. These studies reported a robust elevation of cytokines in brain homogenates after TBI [27].

Reactive astrogliosis is a key component of cellular response to neuroinflammation. Changes in astrocytes were evaluated using an antibody against GFAP, which is a reactive astrocyte marker. Microglia are the main form of adaptive immune response in the central nervous system, which modulates neuronal function during inflammatory responses and developmental synaptic pruning and plasticity in the healthy brain, and can rapidly respond to even minor changes in the brain [28]. In response to harmful stimuli, microglial cells undergo several changes, such as an increase in the number of pro-inflammatory cytokines and the expression of several cell surface antigens [28–30]. Iba-1 is widely used to study microglia because its expression is specific in both reactive and quiescent microglial cells [30].

Our study revealed distinct time-dependent differences in the neuroinflammatory response, using Iba-1 and GFAP, following TBI in both pediatric and adult mice. Pediatric mice exhibited a more early intense increase in neuroinflammatory markers compared with adult mice. Our findings are consistent with those of previous studies that have reported a time-dependent neuroinflammatory response following TBI [31,32]. For example, Fenn et al. [31] reported that diffuse TBI in adult mice promoted neuroinflammation in a time-dependent manner with increased messenger RNA expression of inflammatory mediators (interleukin 1β [IL-1β], tumor necrosis factor α, Iba-1, etc.). Similarly, lzzy et al. [32], who analyzed the temporal course of changes in inflammatory genes of microglia isolated from injured brains at 2, 14, and 60 days after CCI in adult mice, identified a time-dependent, injury-associated changes in microglial gene expression, highlighting a pattern of recovery and transition to a specialized inflammatory state over time.

Few studies exploring time-dependent neuroinflammation after TBI in pediatric populations are available [33–35]. Microgliosis and astrogliosis at 8 days post-TBI in pediatric mice was revealed by increased immunoreactivity for Iba-1 and GFAP in the cortex and hippocampus [33]. In contrast, no significant microglial activation was observed at 1 and 4 DPI. Webster et al. [35] found that the cellular neuroinflammatory response, which involves the activation and migration of astrocytes and microglia, was evident in the brains of pediatric mice at 3 days post-TBI. A strong correlation between neuroinflammation and functional deficits is well-established, particularly in pediatric populations. Arambula et al. [36] and van Erp et al. [37] reported that higher levels of astrocytic and microglial activation were associated with long-term behavioral and cognitive deficits. This aligns with our findings of prolonged GFAP and Iba-1 expression in pediatric mice and suggests that early intervention targeting inflammation could mitigate long-term deficits in pediatric TBI patients. Future studies could examine the potential for anti-inflammatory therapies, such as IL-1β antagonists or microglial modulators, to reduce both short- and long-term neuroinflammation and improve outcomes.

Our study provides several novel insights into the time- and age-dependent neuroinflammatory response following TBI, which distinguishes it from previous research in significant ways. While prior studies primarily focused on either adult or pediatric models, few studies directly compared pediatric and adult responses over short durations. Our research offers a more long-term comparative analysis between pediatric and adult mice. We found specific differences in the duration and intensity of neuroinflammatory responses between the age groups. Detailed time-course comparative analysis revealed that both age groups exhibit a similar neuroinflammatory response to TBI. However, the pediatric brain tends to respond earlier, with a more intense inflammatory response, compared with the adult brain.

Apoptosis is the primary mechanism of cell death. Mitochondria not only participate in caspase-dependent apoptosis but also significantly affect the Bcl-2 pathway during caspase-independent apoptosis [38]. Mitochondrial alterations are one of the main pathways regulating Bcl family proteins, such as Bcl-2, Bax, and caspase-independent apoptosis. Bax is a proapoptotic protein that plays a crucial role in the regulation of apoptosis [39] and is involved in the apoptotic response of neurons and glial cells following TBI [40]. The upregulation of Bax following TBI contributes to neuronal cell death, which can exacerbate damage and hinder recovery. Bcl-2 is an antiapoptotic protein that plays a crucial role in the regulation of apoptosis following TBI by binding to and neutralizing proapoptotic proteins such as Bax and Bcl-2 antagonist/killer (Bak) [41]. By maintaining mitochondrial integrity and preventing the release of apoptogenic factors, Bcl-2 helps preserve neuronal survival and function, which is vital for recovery. Enhancing Bcl-2 activity is a promising therapeutic avenue for reducing neuronal damage and improving outcomes for TBI patients [42]. The Bax/Bcl-2 ratio is therefore a crucial indicator of the apoptotic potential within cells, particularly in TBI. It provides insights into the balance between pro-apoptotic and anti-apoptotic signals, which determines the cell’s fate: survival or death. Following TBI, the Bax/Bcl-2 ratio can provide valuable information about the extent of neuronal injury and the apoptotic response. An increase in the Bax/Bcl-2 ratio after TBI indicates enhanced apoptotic activity, which contributes to neuronal loss and exacerbates brain damage. The more intense apoptotic activity observed in pediatric mice compared with adults is consistent with studies by Andreasson et al. [43] and Ng and Lee [44], who found that developing brains exhibit more pronounced apoptotic responses due to incomplete maturation of protective pathways. This is particularly evident in the Bax/Bcl-2 ratio, with pediatric mice showing a higher propensity for apoptosis. In comparison, adult mice exhibited superior recovery rates of neuronal markers (i.e., NeuN) over time, indicating that the adult brain may be better equipped to handle apoptotic processes. This may explain why pediatric TBI patients often experience more severe cognitive and functional impairments over the long term, as shown by Giza and Hovda [45]. Future studies could examine the potential for anti-inflammatory therapies, such as IL-1β antagonists or microglial modulators, to reduce both short- and long-term neuroinflammation and improve outcomes.

Our study provides a detailed, comparative, temporal analysis of apoptosis in both age groups. While both pediatric and adult mice exhibited peak apoptotic responses, as indicated by the expression of Bax and the Bax/Bcl-2 ratio following TBI at 7 DPI, pediatric mice showed a more intense increase in proapoptotic markers compared with adult mice.

NeuN, also known as RBFOX3, is a neuron-specific nuclear protein widely used to quantify neuronal loss and assess the extent of brain damage in TBI [46]. We used NeuN expression to compare the effects of TBI in pediatric versus adult models, to understand age-dependent differences in neuronal vulnerability and recovery.

In our study, both pediatric and adult mice showed a significant decrease in NeuN expression at 3 DPI, indicating acute neuronal loss post-TBI. The initial neuronal loss was more pronounced in pediatric mice, suggesting a higher sensitivity to injury. Pediatric mice showed sustained reduction in NeuN expression up to 90 DPI, indicating prolonged neurodegeneration and limited recovery over time. The more intense and prolonged reduction in NeuN expression in pediatric mice compared with adult mice suggests that the developing brain is more susceptible to long-term damage following TBI. These findings align with previous research showing that traumatic injury to the immature brain results in progressive neuronal loss, impaired neurogenesis, and hindered recovery [47,48].

HSP70 is a highly conserved protein found in virtually all living organisms and is involved in protecting cells from stress [49]. HSP70 plays a critical role in maintaining protein homeostasis by aiding in the proper folding of nascent and stress-accumulated misfolded proteins, preventing protein aggregation, and assisting in protein transport across cellular membranes [49]. HSP70 protects cells from apoptosis by interfering with key apoptotic signaling pathways. It can inhibit the activation of caspases and prevent the release of cytochrome c from mitochondria, both of which are crucial steps in the apoptotic process. HSP70 has been studied for its neuroprotective effects in TBI [50].

Our study made significant findings regarding the time- and age-dependent expression of HSP70 following TBI in pediatric and adult mice, highlighting the role of HSP70 in the acute stress response to TBI and its potential implications for neuroprotection. Both pediatric and adult mice exhibited a significant increase in HSP70 expression at 7 DPI, indicating a robust stress response. This peak in HSP70 levels suggests that the heat shock response is activated in both age groups as a protective mechanism against injury-induced cellular stress. The return of HSP70 levels to baseline by 90 DPI in both age groups indicates that the acute stress response resolves over time, allowing for potential recovery processes to take place. Given the neuroprotective role of HSP70, enhancing its expression or mimicking its effects could be a viable therapeutic strategy for TBI. Pharmacological agents that induce HSP70 expression or modulate its activity may offer significant benefits in reducing neuronal damage and improving outcomes. Future studies should explore whether prolonged induction of HSP70 through pharmacological agents could provide effective neuroprotection and improve functional outcomes in both pediatric and adult TBI patients.

This study has several limitations. First, we used only male C57BL/6 mice, which may limit the generalizability of the findings. Future studies should include female mice to assess whether similar patterns of neuroinflammation, apoptosis, and HSP70 expression are observed across sexes. The C57BL/6 mouse strain was chosen for this study due to its widespread use in TBI research. However, different mouse strains can exhibit varying degrees of vulnerability and responses to TBI, and our findings may not be directly applicable to other strains. Expanding future research to include multiple strains could provide more comprehensive insights.

Second, we impacted the focal right parietal lobe. While our CCI model is widely used to study TBI, it may not fully replicate diffuse injuries or those affecting other brain regions. Future studies should investigate the effects of diffuse TBI or injuries in different brain regions to compare how the location and type of injury affect neuroinflammation and recovery. Third, although we analyzed neuroinflammatory and apoptotic responses up to 90 DPI, we did not assess chronic changes beyond this period. TBI can result in long-term neuroinflammation and neurodegeneration that may continue beyond 90 days. Future studies should extend the time frame to explore chronic effects and the progression of neurodegeneration. Fourth, while we focused on molecular markers such as GFAP, Iba-1, Bax/Bcl-2 ratio, and HSP70, the study did not assess behavioral or functional outcomes post-TBI. Incorporating neurobehavioral assessments in future studies would provide a more comprehensive understanding of how these molecular changes translate into functional impairments or recovery. Finally, the current study was observational, focusing on the natural course of neuroinflammation and apoptosis after TBI. Investigating potential therapeutic interventions, such as pharmacological agents targeting HSP70 or inflammation, would provide valuable insights into treatment strategies for TBI.

In summary, our study provides novel insights into the age-dependent responses of neuroinflammation, apoptosis, and HSP70 expression following TBI in pediatric and adult mice. Pediatric mice exhibited more intense early neuroinflammatory and apoptotic responses compared with adult mice, highlighting the heightened vulnerability of the developing brain. Both age groups showed a significant but transient increase in HSP70 expression, underscoring its role in the acute stress response. These findings emphasize the need for age-specific therapeutic strategies that effectively mitigate long-term damage and enhance recovery outcomes in TBI patients.