Advancements in traumatic brain injury (TBI) research are driving the emergence of novel therapeutic strategies, especially in light of the lack of curative or disease-modifying treatments for the long-lasting neurological deficits associated with TBI (Naffaa and Naffaa, 2025). As a neurodegenerative condition, TBI is pervaded by widespread neuronal loss mediated through multiple cell death pathways, culminating in discernible pathological behavioral and motor phenotypes (Brett et al., 2021). Among the chief secondary injury mechanisms in TBI are excitotoxicity and oxidative stress (OS), which significantly contribute to DNA damage (Baracaldo-Santamaría et al., 2022; Smith et al., 2013) a crucial factor in promoting neuronal demise by activating various cell death scenarios (Makarevich et al., 2020).

Notably, substantial DNA damage can occur within minutes of the initial injury and persist for days. While intrinsic DNA repair mechanisms are promptly triggered, DNA lesions are frequently observed in TBI (Baracaldo-Santamaría et al., 2022; Smith et al., 2013). Previous studies have shown that DNA fragmentation can persist when the repair machinery is overwhelmed or rendered ineffective by repetitive insults, leading to sustaining functional impairments and progressive neurodegeneration (Davis and Vemuganti, 2021; Welch and Tsai, 2022). This fact underscores that endurance of DNA damage appears to be more pronounced in models of repetitive TBI (RTBI) than in single-impact injuries. Consequently, therapies aimed at minimizing DNA damage and enhancing repair capacity may improve post-TBI recovery outcomes (Davis and Vemuganti, 2021).

Poly (ADP-ribose) polymerase-1 (PARP1) serves as a genomic sentinel, detecting and initiating repair of DNA lesions (Ray Chaudhuri and Nussenzweig, 2017). Nevertheless, hyperactivation of PARP1 unleashes a pathological cascade whereby its product, poly-ADP ribose (PAR), escapes the nucleus, instigating a caspase-independent programmed cell death pathway known as parthanatos, an indispensable player of the pathophysiology of numerous diseases (Fatokun et al., 2014). Extracellular PAR inhibits mitochondrial hexokinase, causing bioenergetic collapse and depleting nicotinamide adenine dinucleotide (NAD⁺) (Andrabi et al., 2014; Fouquerel et al., 2014). In parallel, PAR also interacts with mitochondrial oxidoreductase apoptosis-inducing factor (AIF), prompting its release and translocation to the nucleus (Dawson and Dawson, 2017; Y. Wang et al., 2011). Prior nuclear entry, AIF complexes with macrophage migration inhibitory factor (MIF), and together, they traverse to the nucleus, culminating in the terminal phase of the parthanatos cascade, characterized by genomic DNA fragmentation into segments.

In addition to apoptosis, necroptosis, pyroptosis, and ferroptosis (Dixon et al., 2012; El-Gazar et al., 2024; Raghupathi, 2004), parthanatos represents a unique, NAD⁺ depleting, DNA-damage–driven mode of cell death. This underexplored machinery may be particularly relevant in RTBI, where cumulative oxidative and genotoxic insults persist beyond the acute phase. Although hindering parthanatos trajectory has been proposed as a promising therapeutic avenue for a range of disorders (Wang et al., 2016), including TBI (Sun et al., 2022) its relevance in RTBI has not been addressed, despite the heightened susceptibility of this repeated insult to activate sustained cell death pathways. Potential of this repeated insult to activate sustained cell death signaling pathways.

RTBI, often observed in contact sports and military populations, is characterized by unresolved secondary injury cascades, including persistent oxidative stress and DNA damage (Davis and Vemuganti, 2021; Janković and Pilipović, 2023), which may drive parthanatos activation more robustly than in single-impact models. While prior studies have implicated PARP-1 in TBI (Loane et al., 2015; Zhang et al., 2025), the full parthanatos cascade has yet to be comprehensively explored in the context of RTBI. This is particularly critical, given that such injuries, despite lacking overt initial symptoms, are increasingly recognized as a public health concern due to their contribution to progressive cognitive and behavioral decline and their strong association with neurodegenerative conditions like chronic traumatic encephalopathy (CTE) (Brett et al., 2021; Mckee and Daneshvar, 2015).

Unlike traditional cell-based therapies, extracellular vesicles (EVs) have emerged as promising therapeutic agents for brain disorders due to their ability to cross the blood–brain barrier (BBB), deliver bioactive molecules and enhance intercellular communication (Kumar et al., 2024). Among the various types of EVs, exosomes represent a cell-free therapeutic approach that circumvents the challenges of cell survival, engraftment, and immunogenicity, while retaining robust paracrine regenerative and neuroprotective potential (Huber and Wang, 2023; You and Ikezu, 2019). The modulatory and protective capacities of exosomes largely depend on their biological origin and molecular composition (Kalluri and LeBleu, 2020; J. Wang et al., 2022). In this regard, mesenchymal stem cell (MSC)-derived exosomes have demonstrated wide-ranging therapeutic potential across several disease models by promoting intracellular communication, enhancing tissue regeneration, and mitigating disease progression (Kalluri and LeBleu, 2020; Nikfarjam et al., 2020; Zargar et al., 2022).

Although several recent studies have highlighted the neuroprotective effects of exosomes in TBI, their therapeutic efficacy in the context of RTBI, a condition marked by accumulated redox imbalance and unresolved DNA breakage, remains largely unexplored. Unlike single-impact TBI, RTBI may preferentially engage the caspase-independent, DNA damage–driven cell death pathway parthanatos, amplifying neurodegeneration. In this study, we aim to investigate the neurotherapeutic potential of MSC-derived exosomes in a model of RTBI, with a particular focus on their ability to disrupt parthanatos activation and modulate its downstream molecular cascade. By targeting this underexplored mechanism, the study seeks to establish MSC-derived exosomes as a promising, cell-free intervention for mitigating RTBI-induced neuronal injury.

In line with their neuroprotective potential, the ongoing inquiry highlights for the first time the neurotherapeutic proficiency of MSC-derived exosomes in alleviating RTBI-induced parthanatos-mediated programmed neuronal demise in rats. Through comprehensive analyses, we substantiate that both MSC-derived exosomes regimens comparably fortified the brain against RTBI-evoked neurodegenerative sequelae, as evidenced by the restitution of spatial memory and the conservation of cortical histoarchitecture. In part, these enhancements can be attributed to the inhibition of the triadic neurotoxic nexus; viz., excitotoxicity, OS, and parthanatos cell death. At the molecular level, the treatment regimens dampened cortical glutamate and ROS levels, thereby hindering PARP1 overexpression and the subsequent surge in its downstream effector, PAR, ultimately resulting in NAD + replenishment. Additionally, disrupting this neurotoxic triad declined the protease enzyme calpain, which in turn with the inhibited PAR impeded the release and nuclear translocation of AIF, as well as MIF, thereby obstructing the formation of their deleterious complex that threatens DNA repair. Interestingly, our study confirmed that exosomes treatment restored the two molecular chaperones CYP B, which is studied for the first time in this ailment along with Hsp70, which were bolstered in the untreated RTBI animals, possibly to play a compensatory role against the current detriment. Finally, by thwarting RTBI-induced neurodegenerative cascades, MSC-derived exosomes post-treatment attenuated neuroinflammation, as evidenced by a reduction in cortical immunoreactivity of TNF-α and a diminished IL-1β concentration, further affirming the anti-inflammatory potency of MSC-derived exosomes.

In evaluating the physicochemical characteristics of exosomes, it is important to consider methodological differences in size measurements. Discrepancy between TEM and DLS is not uncommon; TEM provides a snapshot of vesicle morphology under dehydrated conditions, often yielding smaller sizes, while DLS measures the hydrodynamic diameter in solution, which may appear larger due to the presence of surface-bound proteins or other biomolecules. A consistent size difference of approximately 40 nm between DLS and TEM has been reported (Lyu et al., 2021), and similar findings have been documented in other studies (Chernyshev et al., 2015; Sokolova et al., 2011), reinforcing that such differences stem from the techniques used rather than variations in exosome quality.

The observed phenotype, CD45^-/CD9⁺/CD63⁺, indicates a pure population of isolated exosomes. CD9 and CD63, members of the tetraspanin family found on the exosomal surface, are not directly therapeutic themselves but play a pivotal role in mediating the therapeutic potential of exosomes. Their expression is associated with key functions such as cellular targeting, exosome stability, and immune modulation. These features make them vital for exosome-based interventions in neurodegenerative disorders, including TBI. Accordingly, exosomes enriched with the identified CD markers can serve as carriers for neuroprotective cargos, including specific microRNAs, various proteins, and anti-inflammatory cytokines, capable of crossing the BBB to target affected neurons (Dehghani et al., 2025; Jankovičová et al., 2020). This targeted delivery may impede disease progression by attenuating inflammation and enhancing neuronal survival in neurodegenerative conditions (Nouri et al., 2024; Zhang et al., 2024).

In the current work, the NORT substantiated the therapeutic capacity of MSC-derived exosomes in alleviating RTBI-induced aberrant behavioral response, which diminished exploratory proclivities and engagement with novel objects corroborating previous observations in various neurological disorders (Fallahi et al., 2024; Jovanovic et al., 2018). The memory restitution prowess of MSC-derived exosomes therapy was further validated by the Y-maze results reported herein. The behavioral anomalies alongside the changes in cortical architecture depicted in the RTBI-injured animals, may be ascribed not only to the primary mechanical trauma from the weight drop, resulting in disrupted brain architecture, but also to the second sequelae of trauma. Hence, from a biomechanical perspective, RTBI was associated with a pronounced surge in the neurotoxin glutamate and aggravated oxidative duress, phenomena validated in the present study and corroborated by prior studies (Mckee and Daneshvar, 2015) playing a cardinal role in precipitating subsequent deleterious cascades.

Accordingly, the neurotherapeutic capacity of MSC-derived exosomes regimens to ameliorate behavioral and histopathological deficits can be partially accredited to their ability to diminish cortical glutamate content, reinforcing an earlier observation involving MSC-driven exosomes in mild single-impact brain trauma (Zhuang et al., 2022). Despite the renowned nexus between OS and RTBI pathophysiology (El-Gazar et al., 2024; Zink et al., 2010), no prior evidence has chronicled the antioxidant prowess of MSC-driven exosomes in repetitive TBI models. Nevertheless, their competency in mitigating OS has been reported in alternative central nervous system injury paradigms (Quan et al., 2025; Xie et al., 2023; Zaazaa et al., 2021), where they contributed to cognitive enhancement, spatial memory consolidation, and augmented exploratory behavior. This reparative aptitude of exosomes is largely attributed to their potent intercellular signaling capabilities and their cargo of bioactive mediators (Y. Chen et al., 2020; Reza-Zaldivar et al., 2019).

The TBI-induced free radicals (FRs) generation and ROS overproduction is mediated primarily via mitochondrial disruption (Besson, 2009). This oxidative assault damages cellular components, including DNA, lipids, and proteins. Substantial DNA cleavage has been reported post-TBI (Davis and Vemuganti, 2021; LaPlaca et al., 1999; Schwab, Tator and Hazrati, 2019), leading to the activation of PARP1 as a reparative attempt. However, under excessive genotoxic stress, PARP1 becomes pathologically hyperactivated, exacerbating cellular damage (Besson, 2009; Huang et al., 2022). This dysregulation has been associated with cognitive impairments observed both in this study and in previous TBI models, where PARP1 inactivation alleviated such deficits (Whalen et al., 1999).

The neuroprotective efficacy of MSC-derived exosomes primarily hinges on the marked downregulation of PARP1 overexpression, an adjustment that was paralleled by a considerable increment in NAD⁺ levels to aid in mitigating neurodegeneration. In fact, NAD⁺ serves as a crucial redox cofactor indispensable for preserving mitochondrial bioenergetics by fine-tuning the generation and scavenging of ROS. Moreover, NAD⁺ orchestrates an array of essential cellular processes, including DNA repair, metabolic equilibrium, cell cycle regulation, protein-protein signaling, and modulation of inflammatory cascades (Martínez-Morcillo et al., 2021). As reflected in our findings, RTBI-provoked upregulation of PARP1 culminated in NAD⁺ attrition, a cardinal event in the parthanatos paradigm, wherein excessive NAD⁺ consumption during abortive DNA repair triggers energetic collapse and exacerbates mitochondrial impairment that propels cell demise (Murata et al., 2019). In tandem, NAD⁺ paucity contributes to unchecked ROS accumulation, perpetuating a vicious cycle of oxidative damage (Ying, 2013). In support, previous studies have documented PARP-driven NAD⁺ exhaustion in hippocampal and cortical regions post-trauma (Y. Lai et al., 2008) to brace our current data.

The function of the PARP1 enzyme also governs PAR catabolism and must be meticulously regulated to prevent either excessive PAR buildup or untimely PAR breakdown. However, in case of hyperactivated PARP1 and energy depletion, the metabolically exhaustive cycle is catalyzed, generating an overabundant PAR polymer that translocates to cytoplasm and mitochondria, where it interacts with AIF, triggering its release (Y. Wang et al., 2011). In the cytoplasm, AIF engages in a molecular liaison with the nuclease protein MIF, enlisting it as a nuclear chaperone to facilitate its translocation into the nucleus, where MIF slices genomic DNA into substantial segments and exacerbates cellular damage (Huang et al., 2022)As a result of the thwarted glutamate/ROS/PARP1, and the replenishment of NAD+, MSC-derived exosomes therapy, restored cortical content of PAR, as well as the nuclear level of both AIF and MIF, pointing for the possible DNA repair. In concordance with the significant ablation of the AIF and MIF deleterious molecules, Wang et al. (2016) demonstrated that the genetic abrogation of MIF or perturbation of the AIF-MIF intricate nexus nullified its endonuclease aptitude, thereby impeding glutamate-induced excitotoxic neuronal demise in a focal cerebral ischemia model.

In our study, MSC-derived exosomes effectively suppressed elevated cortical calpain levels, aiding in the restoration of cellular homeostasis. Beyond glutamate-induced Ca²⁺-driven FRs generation, recent evidence suggests that Ca²⁺ signaling also activates PARP1 (Zhou et al., 2021), partly via Ca²⁺-dependent proteases like calpains (Besson, 2009), a finding consistent with our RTBI model. In fact, activated calpain contributes significantly to neuronal injury by degrading key neuronal proteins (Metwally et al., 2023), disrupting the nuclear envelope (Singh et al., 2024), and cleaving mitochondrial-protective proteins, thus promoting mitochondrial dysfunction (Shan et al., 2022). It also facilitates AIF maturation and its mitochondrial egress (Cao et al., 2007). However, whether calpain inhibition genuinely enhances cell survival in PARP1-driven cell death or merely delays AIF translocation remains uncertain (J. Zhang et al., 2025). Regardless, MSC-derived exosomes mediated inhibition of calpain likely contributed to attenuating parthanatos-associated neurodegeneration.

Our study assessed for the first time the potential impact of RTBI and MSC-derived exosomes on the molecular chaperone CYP B. Our findings showed that treatment with MSC-derived exosomes succeeded in restoring CYP B to its normal level after being boosted in the RTBI model. Indeed, CYPs constitute a subset of immunophilin proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPIase) catalytic functionality and are implicated in diverse cellular processes, including protein folding, signal transduction, intracellular trafficking, mitochondrial preservation, and transcriptional regulation (Andreeva et al., 1999; Gegunde et al., 2021; Liang et al., 2021). The CYP B chaperon is distinctive due to possessing an endoplasmic reticulum (ER)-targeting signal sequence, though it is also present in the extracellular space and the nucleus (Oh et al., 2011). Despite the scarce data on the role of CYP B in neurodegenerative diseases, an earlier in-vitro study pointed to the ability of overexpressed CYP B to attenuate PARP cleavage, DNA disintegration, and ROS-induced neurotoxicity mediated by beta amyloid 25–35 model (Oh et al., 2011). Subsequently, in an Alzheimer’s disease paradigm, the investigators disclosed that suppression of the ER-resident chaperone, CYP B, instigates presenilin accrual concomitant with mitochondrial perturbation (Ben-Gedalya et al., 2015). Concordant with the neuroprotective capacity attributed to CYP B, Zhuang et al. (Zhuang et al., 2022) reported its involvement in the intracellular internalization of nucleic acids. Given the salutary attributes of CYP B, we posited that its RTBI-induced stimulation may serve a compensatory function counteracting the neurodegenerative sequelae of RTBI. Supporting this premise, Wang et al. (B. Wang et al., 2016) documented that CYP B overexpression ameliorated aldosterone-evoked nephrotoxicity by enhancing mitochondrial performance and curbing ROS accumulation. Intriguingly, and to support our findings, these authors reported bolstered levels of the molecular chaperone in the aldosterone-induced kidney injury model, implying a compensatory response in renal tubular epithelia. Since MSC-derived exosomes were reported to reduce protein misfolding and enhance mitochondria function indeed by its current aptitude to preserve NAD⁺ with its critical anti-parthanatos efficacy could be the culprits for the reduced CYP B cortical content observed in our study.

Another molecular chaperone that was boosted in RTBI cohort is Hsp70 that governs proteostasis by balancing protein proper folding, functional regulation, and catabolism to mediate its chaperone therapeutic impact (Kim et al., 2020). Being a stress-inducible effector, Hsp70 provokes its survival potential across diverse pathological milieus, including tissue injury, ischemia-reperfusion insult, and inflammatory states (Zhao et al., 2013). Additionally, Hsp70 cytoprotective actions are enacted through attenuating intrinsic apoptotic signaling, preserving mitochondrial integrity (Tsuchiya et al., 2003), stifling Ca²⁺ intracellular accumulation in a model of ischemia/reoxygenation (Liu et al., 2016) hampering thus cellular injury. These effects can partake in the Hsp70 anti-parthanatos capacity, besides its ability to interlock AIF, thereby restraining its nuclear translocation (Matsumori et al., 2005). Furthermore, Hsp70 tempers caspase-independent demise, where it was found to exhibit nuclear and nucleolar co-localization with PARP1, a molecular interplay that implies a synergistic involvement in orchestrating DNA repair pathways and mediating cellular adaptive responses under genotoxic or stress-inducing conditions (Kotoglou et al., 2009). To further signify its neuroprotective effect, exogenous augmentation of Hsp70 expression has been shown to diminish neuronal attrition in experimental paradigms of cerebral ischemia and in kainic acid-induced excitotoxicity. In contrast, Hsp70-deficient murine models exhibit exacerbated cerebral infarction post-ischemia (Lee et al., 2001). In such contexts, the overexpressed Hsp70-mediated orchestration of key neurodegenerative cascades, may rationalize its heightened levels in stress, injured animals as a recuperative mechanism, supplementing the primary molecular chaperone CYP B.

Interestingly, treatment with MSC-derived exosomes attenuated the elevated levels of Hsp70 and CYP B observed in RTBI-exposed animals. This reduction may reflect a diminished cellular burden, potentially lowering the need for compensatory upregulation of these stress-related chaperones. However, the precise functional roles of Hsp70 and CYP B in the context of RTBI remain to be fully elucidated, and further studies are warranted to confirm this interpretation. In support of their potential cytoprotective capacity, MSC-derived exosomes are known to deliver functional proteasomes, including the 20S catalytic core, to injured tissues (R. C. Lai et al., 2012). This proteolytic complex plays a key role in maintaining proteostasis by degrading misfolded or aggregated proteins, thereby alleviating cellular stress (Pepelnjak et al., 2024; Yeh Yeo et al., 2013). Moreover, MSC-derived exosomes possess well-documented antioxidant (Elnegris et al., 2024), anti-inflammatory (Chen et al., 2024), and antiapoptotic (Zhuang et al., 2023) properties, which may collectively contribute to reducing the reliance on chaperone-mediated stress responses following RTBI.

Additionally, MSC-derived exosomes underscored their anti-inflammatory effect by curbing the protein expression of both TNF-α and IL-1β to align with an earlier study using MSC-derived exosomes in early-stage post single hit (Ni et al., 2019). In a recent study, Tang et al. (Tang et al., 2023) chronicled that exosomes derived from adipose-derived stem cells were able to quell both inflammatory markers in microglia exposed to lipopolysaccharide. RTBI-induced inflammation is the final detrimental pivot recorded herein to exacerbate neuronal injury and compromising neural repair mechanisms. This event has been documented by previous studies (Hiskens et al., 2021; J. Wang et al., 2025). Moreover, exosomes-mediated inhibition of the parthanatos axis further amplifies their robust anti-inflammatory efficacy. In this vein, PARP1 antagonists have been shown to markedly suppress inflammatory indices in TBI rat paradigms (d’Avila et al., 2012) and in a model of localized aseptic meningitis (Rom et al., 2016). Similarly, neuroinflammation was attenuated in a murine TBI model following administration of a parthanatos modulator (Shi et al., 2022) or calpain suppressor (Tao et al., 2017). A prior investigation also underscored the anti-inflammatory utility of NAD⁺ in shielding neural tissue subjected to TBI and ischemic insults (Ying, 2013).

This study unveils a promising therapeutic paradigm where MSC-derived exosomes confer robust neuroprotection against the multifaceted damage of RTBI. Through broad-spectrum modulation of parthanatos-induced cell death, and the secondary injurious mechanisms excitotoxicity, oxidative duress, and inflammation, MSC-derived exosomes significantly restored cognitive performance and preserved cortical structure. Mechanistically, they curbed glutamate and ROS levels, halting the parthanatos cascade by inhibiting PARP1, PAR, and the nuclear translocation of AIF and its nuclease partner MIF. Additionally, calpain suppression added a vital layer of mitochondrial and nuclear safeguarding. Intriguingly, MSC-derived exosomes rebalanced the heightened expression of CYP B and Hsp70, stress-responsive chaperones likely upregulated as part of an intrinsic compensatory defense response. These findings highlight the involvement of parthanatos in RTBI-associated neurodegeneration and provide a strong preclinical foundation for the translational application of MSC-derived exosomes as a novel therapeutic strategy in managing RTBI-associated neurodegeneration. Future research should refine delivery protocols and evaluate the potential of MSC-derived exosomes to influence long-term outcomes of RTBI, including chronic neurodegenerative changes, thereby helping to bridge the gap between bench and bedside.

Despite the encouraging results, this study has several limitations. The relatively short follow-up period limits conclusions regarding the long-term efficacy and safety of MSC-derived exosomes in RTBI. Longitudinal studies are needed to assess the durability of neuroprotection and explore the potential of repeated dosing to prevent or delay chronic neurodegenerative outcomes. Additionally, the exclusive use of male Wistar rats restricts the generalizability of the findings across sexes and species. Furthermore, the behavioral assessment incorporating a broader battery of cognitive and motor evaluations would offer a more comprehensive view of the therapeutic impact. These considerations underscore the need for further research to validate and extend the current findings.

Future studies should explore the long-term efficacy of MSC-derived exosomes in RTBI, particularly their potential to prevent chronic neurodegenerative outcomes such as tau and TDP-43 pathology. Investigating the role of parthanatos in chronic injury, optimizing dosing strategies, and evaluating alternative delivery routes will be essential for clinical translation. Standardization of human exosome preparations and comprehensive safety assessments, including immunogenicity and tissue distribution, will further enhance the therapeutic viability of this cell-free approach.