The long-term influences of age at injury on neuroinflammation and neuronal apoptosis following traumatic brain injury in pediatric and adult mice
Article information
Abstract
Objective
The study explores the long-term impacts of traumatic brain injury (TBI) on neuroinflammation and neuronal apoptosis in pediatric and adult mice, focusing on how age at injury influences these processes.
Methods
Controlled cortical impacts were used to induce TBI in pediatric (21–25 days old) and adult (8–12 weeks old) C57BL/6 male mice. Neuroinflammation was evaluated by measuring immunoreactivity for allograft inflammatory factor 1 (AIF-1)/ionized calcium-binding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP), while apoptosis was assessed using markers such as B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, and procaspase-3. Additionally, heat shock protein 70 (HSP70) expression was measured to understand the stress response.
Results
Following controlled cortical impacts, pediatric mice exhibited a significant reduction in expression of neuronal nuclei (P<0.001), and significant increases in expression of GFAP (P<0.01) and AIF-1/Iba-1 (P<0.05) at 3 days post-injury (DPI) compared with sham controls. In contrast, adult mice exhibited no significant change in AIF-1/Iba-1 expression and a less pronounced increase in GFAP (P<0.05) at 3 DPI compared with sham controls. A more significant increase in Bax/Bcl-2 ratio at 7 DPI (P<0.01) was seen in pediatric mice, while a weak but significant increase in Bax/Bcl-2 ratio at 7 DPI (P<0.05) was evident in adults. Both age groups showed a significant but transient increase in HSP70 levels at 7 DPI, which normalized by 90 DPI.
Conclusion
Pediatric and adult mice exhibited significant time-dependent differences in neuroinflammation and apoptosis following TBI, with pediatric mice showing more intense early responses indicative of age-specific vulnerabilities in post-injury outcomes. Both age groups showed a significant but transient increase in HSP70 expression, suggesting an acute response to stress post-injury.
INTRODUCTION
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.
METHODS
Ethics statement
All experimental protocols were approved by the Animal Care and Use Committee of Chungbuk National University (No. CBNUR-854-15). The study was conducted in accordance with the ethical guidelines for animal research.
Animal population
Pediatric (21–25 days old) and adult (8–12 weeks old) C57BL/6 male mice were used in this study. The mice were housed under a standard 12-hour light/dark cycle with ad libitum access to food and water.
Controlled cortical impact
CCI was performed as previously described [25,26]. Briefly, mice were anesthetized with an intramuscular injection of tiletamine/zolazepam (Zoletil, 15 mg/kg; Virbac). After shaving and cleaning the area of the head between the ears with betadine, a midline scalp incision was made to expose the right parietal bone. The surgical landmarks used for craniotomy were further detailed, including the specific location between the lambda and bregma 0.5 mm from the midline. A circle 4 mm in diameter was then drawn in the center of the lambda and bregma 0.5 mm from the midline. A right parietal craniotomy was carefully performed along the marked circle using a surgical microscope and micromotor drill (Stoelting). The CCI device was calibrated with respect to the exposed dura mater during the craniotomy. The impactor calibration process for the injured animals was specified, ensuring a depth of 2.0 mm, a velocity of 3.0 m/sec, and a duration of 500 milliseconds were maintained consistently across all experimental groups. After the impact, the scalp incision was sutured with 5-0 nylon. The animals were then returned to their cages. A sham-operated group underwent craniotomy without CCI injury. CCI mice used for western blotting were sacrificed at days 3, 7, and 90 after CCI. The surgical procedure is illustrated in Fig. 1.
Experimental schedule. The timeline details the schedule from baseline to 90 days post-injury (DPI). Pediatric and adult mice underwent controlled cortical impact (CCI), with sacrifices and biochemical studies conducted at 3, 7, and 90 DPI. Assessments focused on neuroinflammation, apoptosis, and cell stress. (A, B) The procedure for CCI. At the beginning of surgery (day 0), the mouse head was stably fixed on the stereotactic frame with ear bar and mouth bits. (A) The right skull was exposed, and a 4-mm circle was drawn in the center of bregma and lambda. (B) The bone was removed by drilling to generate a window for impact. The impactor tip was retracted and lowered to the surface of the exposed dura until contact was made.
Western blotting
At 3, 7, and 90 days post-injury (DPI), the mice were sedated with an intramuscular injection of 15 mg/kg tiletamine/zolazepam and sacrificed. Brain tissue (3–5 in each group) was dissected and stored at −80 ℃ immediately before use. The samples were lysed with an ice-cold radioimmunoprecipitation assay (RIPA) buffer (#MB-030-0050, Rockland) supplemented with a 10 μL/mL protease and phosphatase dual-inhibitor cocktail (#P3300-001, GenDEPOT) and homogenized using a syringe with a 23-gauge needle (approximately 10 to 20 times) at room temperature (RT). The tissue lysates were incubated on ice for 30 minutes and mixed using a vortex every 5 minutes. The supernatants were collected after centrifugation at 13,000 rpm at 4 ℃ for 10 minutes.
A Bradford assay (#5000202, Bio-Rad Laboratories) was used to measure protein concentration. Each sample was then treated with a 4× Laemmli sample buffer (#161-0747, Bio-Rad Laboratories) containing 10% (volume/volume) β-mercaptoethanol (#M3148, Sigma-Aldrich). Samples were boiled in a dry bath at 95 ℃ for 10 minutes, stabilized on ice for 5 minutes, and then centrifuged at 13,000 rpm and 4 ℃ for 10 minutes. The supernatants (10 μg in 4, 7.8, 10, or 13.4 μL) were separated using Any kD Mini-PROTEAN TGX Precast Protein Gels (#456-9034, Bio-Rad Laboratories), subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (#165-8004, Bio-Rad Laboratories) at a constant voltage of 200 V for 35 minutes. Proteins were transferred to a hydrophobic bond polyvinylidene fluoride (PVDF) transfer membrane with a pore size of 0.45 μm (#10600023, Amersham) or 0.2 μm (#10600021, Amersham) using the wet-tank transfer method (#TE22, Hoefer Inc) at 250 mA for 60 minutes.
Membranes were incubated with 15 mL of 5% (weight/volume, w/v) bovine serum albumin (#A0100-010, GenDEPOT) in Tris-buffered saline with Tween 20 (TBS-T) and 0.1% Tween-20 (#274348, Sigma-Aldrich) in 1×TBS buffer (#TR2008-100-00, Biosesang) to block nonspecific reactions for 2 hours at RT using a laboratory shaker (20 rpm; #AD-ST, Gyrozen) and washed three times with 15 mL of TBS-T for 10 minutes at RT (40 rpm). Membranes were then incubated overnight at 4 ℃ with 10 mL of 5% (w/v) bovine serum albumin (BSA) in TBS-T containing an appropriate concentration of the primary antibodies against RNA-binding protein, including fox-1 homology 3 (RBFOX3)/neuron-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), allograft inflammatory factor 1(AIF-1)/ionized calcium-binding adapter molecule 1 (Iba-1), B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, procaspase-3, and β-actin for 18 to 24 hours, and then rinsed three time with 15 mL of TBS-T for 10 minutes at RT. After the second washing, the membranes were incubated with 15 mL of 5% (w/v) BSA in TBS-T, including horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:3,000; #170-6515, Bio-Rad Laboratories) or goat anti-mouse (1:3,000; #170-6516; Bio-Rad Laboratories) polyclonal antibodies for 1 hour at RT (20 rpm), and then washed three times with 15 mL of TBS-T for 10 minutes at RT. After the third washing, the bands were visualized using a Clarity western enhanced chemiluminescence substrate (#170-5060, Bio-Rad Laboratories) and a ChemiDoc XRS+ Imaging System (#170-8265, Bio-Rad Laboratories) in accordance with the manufacturer’s protocol. Protein bands were then analyzed quantitatively in ImageJ ver. 1.53k (US National Institutes of Health), and the results were used for further statistical analysis. Expression levels of the target protein in the cortex of the mice were determined relative to β-actin as an internal loading control, and all relative band intensities of target protein were normalized to the mean relative band intensity of the control and sham groups.
Statistical analysis
All data are presented as the mean±standard error of the mean. To ensure data accuracy, all western blot studies were repeated three to six times. GraphPad Prism ver. 9.3.1 (GraphPad Software) was used to analyze the normalized data and construct histograms. Data normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was confirmed using the Brown-Forsythe test. If the data did not meet these assumptions, appropriate nonparametric tests, such as the Kruskal-Wallis test followed by Dunn post hoc test, were used to ensure robust results. A two-way analysis of variance was used to analyze the effects of age at injury (sham vs. TBI). When significant interactions were observed, post hoc comparisons were conducted using Tukey post hoc test for equal sample sizes or Bonferroni post hoc test for unequal sample sizes. All significant differences were interpreted in the context of P-values (e.g., P<0.05, P<0.01, P<0.001).
RESULTS
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.
Survival and weight gain after CCI
The survival rate in pediatric mice following CCI was significantly lower than in adult mice. Pediatric 2-mm CCI mice experienced a notable decline in survival, stabilizing at approximately 50% by 90 DPI, whereas adult 2-mm CCI mice showed a slight decrease in survival, maintaining approximately 80% survival by 90 DPI (Fig. 2A). While the adult sham group maintained a 100% survival rate throughout the experiment, the pediatric 2-mm TBI group showed a substantial decline. Weight gain was observed in all groups post-injury, although both the pediatric and adult 2-mm CCI groups exhibited slightly reduced weight gain compared with their respective sham groups (Fig. 2B).
Percent survival and weight gain over 90 days following traumatic brain injury (TBI) in pediatric and adult mice. (A) Adult sham group shows 100% survival. The adult 2-mm TBI group shows a slight survival decrease, while the pediatric 2-mm TBI group shows a significant survival decrease, stabilizing near 50% by 90 days. (B) Both pediatric sham and 2-mm TBI groups show significant weight gain, with slightly reduced weight gain. The adult 2-mm TBI group shows slightly reduced weight gain compared with the adult sham group.
Time-dependent neuronal cell survival following CCI in pediatric and adult mice
Expression of NeuN, a marker of neuronal cell survival, was significantly reduced following CCI in both pediatric and adult mice. Pediatric mice exhibited a substantial reduction in NeuN expression as early as 3 days DPI (P<0.001) and remained significantly lower at 7 DPI (P<0.01) and 90 DPI (P<0.05) compared with sham controls (Fig. 3B). This reduction indicates a sustained loss of neuronal cells. Conversely, while adult mice also showed significant NeuN reduction at 3 DPI (P<0.001), they demonstrated partial recovery by 90 DPI, with NeuN levels approaching those of the sham group (Fig. 4B), suggesting some degree of neuronal recovery over time.
Time-dependent expression of neuroinflammatory and neuronal markers in pediatric mice. (A) Western blot bands and (B–D) densitometry analysis show the expression levels of RNA-binding protein, including fox-1 homology 3 (RBFOX3)/neuron-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), and allograft inflammatory factor 1 (AIF-1)/ionized calcium-binding adapter molecule 1 (Iba-1) at 3, 7, and 90 days post-injury (DPI) in both sham and 2-mm traumatic brain injury (TBI) groups. The β-actin was used as the internal loading control. All data are presented as mean±standard error of the mean, and each experiment was repeated more than three times (four or five times in each group). Statistical significance was analyzed by two-way analysis of variance (ANOVA). When an interaction effect occurred (P<0.05 shown by ANOVA), a Tukey or Bonferroni post hoc test was used to assess the difference between groups. *P<0.05, **P<0.01, and ***P<0.001.
Time-dependent expression of neuroinflammatory and neuronal markers in adult mice. (A) Western blot bands and (B–D) densitometry analysis show the expression levels of RNA-binding protein, including fox-1 homology 3 (RBFOX3)/neuron-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), and allograft inflammatory factor 1 (AIF-1)/ionized calcium-binding adapter molecule 1 (Iba-1) at 3, 7, and 90 days post-injury (DPI) in both sham and 2-mm traumatic brain injury (TBI) groups. The β-actin was used as the internal loading control. All data are presented as the mean±standard error of the mean, and each experiment was repeated more than three times (four or five times in each group). Statistical significance was analyzed by two-way analysis of variance (ANOVA). When an interaction effect occurred (P<0.05 shown by ANOVA), a Tukey or Bonferroni post hoc test was used to assess the difference between groups. *P<0.05, **P<0.01, and ***P<0.001.
Time-dependent neuroinflammation following CCI in pediatric and adult mice
Following CCI, pediatric mice exhibited a significantly stronger and earlier neuroinflammatory response compared with adults. GFAP expression, which is indicative of astrocyte activation, was markedly elevated in pediatric mice at 3 DPI, peaking at 7 DPI (P<0.001), and remaining significantly elevated at 90 DPI compared with sham controls (Fig. 3C). Similarly, expression of AIF-1/Iba-1 expression, a marker of microglial activation, increased in pediatric mice at 3 DPI (P<0.05), peaking at 7 DPI (P<0.01), and subsequently decreasing at 90 DPI (Fig. 3D). These findings suggest an early and sustained neuroinflammatory response in the pediatric brain.
In adult mice, the neuroinflammatory response followed a similar time-dependent pattern but was less pronounced. AIF-1/Iba-1 expression peaked at 7 DPI (P<0.001) before returning to near-baseline levels by 90 DPI (Fig. 4D). Unlike adults, pediatric mice showed significantly elevated AIF-1/Iba-1 expression as early as 3 DPI (P<0.05), indicating heightened early microglial response to injury (Fig. 3D).
Time-dependent apoptosis following CCI in pediatric and adult mice
Both pediatric and adult mice exhibited time-dependent increases in proapoptotic markers post-CCI, with pediatric mice showing a more intense apoptotic response. In pediatric mice, Bax expression increased by a factor of 2.48 at 7 DPI (P<0.01) compared with sham controls, accompanied by a significant rise in the Bax/Bcl-2 ratio, suggesting an enhanced apoptotic response at early stages (Fig. 5). Adult mice exhibited a milder increase in Bax expression, 1.82 times at 7 DPI (P<0.05), and a more moderate rise in the Bax/Bcl-2 ratio (P<0.05), indicating a less pronounced apoptotic response (Fig. 6).
Time-dependent expression of apoptosis markers in pediatric mice. (A) Western blot bands and (B–E) densitometry analysis show the expression levels of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, and procaspase-3 at 3, 7, and 90 days post-injury (DPI) in both the sham and 2-mm traumatic brain injury (TBI) groups. The β-actin was used as the internal loading control. All data are presented as the mean±standard error of the mean, and each experiment was repeated more than three times (four or five times in each group). Statistical significance was analyzed by two-way analysis of variance (ANOVA). When an interaction effect occurred (P<0.05 shown by ANOVA), a Tukey or Bonferroni post hoc test was used to assess the difference between groups. **P<0.01.
Time-dependent expression of apoptosis markers in adult mice. (A) Western blot bands and (B–E) densitometry analysis show the expression levels of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, and procaspase-3 at 3, 7, and 90 days post-injury (DPI) in both the sham and 2-mm traumatic brain injury (TBI) groups. The β-actin was used as the internal loading control. All data are presented as the mean±standard error of the mean, and each experiment was repeated more than three times (four or five times in each group). Statistical significance was analyzed by two-way analysis of variance (ANOVA). When an interaction effect occurred (P<0.05 shown by ANOVA), a Tukey or Bonferroni post hoc test was used to assess the difference between groups. *P<0.05.
Time-dependent HSP70 expression following CCI in pediatric and adult mice
Both pediatric and adult mice showed a significant, transient increase in HSP70 expression at 7 DPI, indicative of a robust acute stress response. In pediatric mice, HSP70 levels were 1.55 times greater than those in sham controls (P<0.05), while in adult mice, HSP70 expression increased by a factor of 1.36 (P<0.05) (Fig. 7). By 90 DPI, HSP70 levels had returned to baseline in both groups, indicating resolution of the acute stress response.
Time-dependent expression of heat shock protein 70 (HSP70) in adult and pediatric mice. (A, B) Western blot bands and (C, D) densitometry analysis show the expression levels of at 3, 7, and 90 days post-injury (DPI) in both the sham and 2-mm traumatic brain injury (TBI) groups. The β-actin was used as the internal loading control. All data are presented as the mean±standard error of the mean, and each experiment was repeated more than three times (four or five times in each group). Statistical significance was analyzed by two-way analysis of variance (ANOVA). When an interaction effect occurred (P<0.05 shown by ANOVA), a Tukey or Bonferroni post hoc test was used to assess the difference between groups. *P<0.05.
DISCUSSION
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.
Notes
Author contributions
Conceptualization: HK, SWL; Data curation: JSP, HJP, YMK; Formal analysis: HSC, GGY; Funding acquisition: HK; Investigation: JSP, GJP, SCK; Methodology: SWL, HSC; Project administration: HK; Resources: SCK, SWL; Software: GGY, HSC; Supervision: HK; Validation: YMK, GJP; Visualization: HJP, YMK; Writing–original draft: JSP, HJP; Writing–review & editing: all authors. All authors read and approved the final manuscript.
Conflicts of interest
The authors have no conflicts of interest to declare.
Funding
This study was supported by the National Research Foundation of Korea (NRF) grant (No. RS-2024-00358977), funded by the Korean Ministry of Science and ICT.
Data availability
Data analyzed in this study are available from the corresponding author upon reasonable request.
References
Article information Continued
Notes
Capsule Summary
What is already known
Neuroinflammation is a well-established component of both acute and chronic injuries following traumatic brain injury (TBI). Apoptosis contributes to neuronal loss and functional deficits following TBI. 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.
What is new in the current study
Pediatric and adult mice showed significant time-dependent differences in the severity of neuroinflammation and apoptosis, following a similar overall pattern. Pediatric mice exhibited more early intense inflammatory response and apoptotic activities compared to adult mice. Both age groups showed a significant but transient increase in HSP70 expression, suggesting an acute stress response post-injury.