A traumatic brain injury (TBI) is often caused by a blunt head trauma event significantly affecting the quality of life and health of an individual. In some cases, a TBI can even threaten a patient’s life, as it has been shown that TBI is associated with increased morbidity and mortality rates worldwide. Mild TBI (mTBI), also known as a concussion, accounts for approximately 80% of all TBI cases [1]. Despite the generally favorable outcomes for most mTBI patients, some individuals continue to experience chronic post-concussion (PC) symptoms, including cognitive impairment, headaches, sleep disturbances, and mood disorders, which can significantly impact their quality of life [2]. Early diagnosis and management of mTBI and its associated post-concussion syndrome (PCS) are crucial to prevent long-term complications. However, the current diagnostic methods for mTBI and PCS are limited and often rely on subjective clinical evaluations [2].

Exosomes are small extracellular vesicles released by almost all mammalian cell types, including neurons and glial cells [3]. Neuron-derived exosomes are extracellular vesicles released by neurons. They are thought to be key mediators in communication and waste management within brain tissues [4]. The diameters of exosomes vary between 30 and 150 nm [5]. Exosomes were first described in the 1980s [6]. They were thought to originate from the endomembrane system, while their membranous envelope is invaginated during the maturation process and forms the intraluminal vesicles (ILVs).

The ILVs consist of proteins, nucleic acids, and lipids. Mature endosomes that contain numerous ILVs are called multivesicular bodies (MVB) [7]. Multivesicular bodies are either degraded by lysosomes or transported to cell membranes; fuse with the cell membranes; and release the inner vesicles into the extracellular space, forming exosomes loaded with proteins, non-coding RNAs, lipids, and other active substances [8]. They contain particular and varied types of markers that contribute to identifying their origins. Once they are secreted, they can be internalized by recipient cells through different mechanisms, such as phagocytosis, micropinocytosis, endocytosis, and plasma membrane fusion [9,10,11]. Neuron- and glial-derived exosomes carry and release multiple molecules related to neuronal function and neurotransmission in the brain. They are essential in neuronal development, neuroimmune communication, and synaptic spasticity [11].

The roles of exosomes and the changes in the exosomal content in TBI have been extensively investigated over the past few years. Changes in the levels of exosomal content after a TBI can assist in the diagnosis and severity classification of the TBI in question [12]. The concentration of neuro-derived exosomes in the plasma of patients with an mTBI is reduced by 45% in the acute phase of the injury, while alterations in the levels of neuropathological protein in these exosomes can reveal phase and severity specificity [13].

Recent studies have shown promising results with respect to using salivary exosomal biomarkers as potential diagnostic and prognostic tools for mTBI and PCS. Also, the importance of exosomes was previously described in relation to mTBI and PCS, and studies have shown that their detection could be significantly correlated with the time passed after a traumatic event and the extent of the subsequent damage [32]. Naturally, the role of exosomes within the brain tissues is to mediate intercellular communication [33]. However, in cases of a crisis, exosomes were found to act as potent mediators of neuronal response to stress, inflammation, and regeneration [34,35]. Furthermore, exosomes are potent molecular complexes that can be successfully isolated or even synthetized in vitro and used as potential therapeutic agents [36]. They can, moreover, be efficient carriers of active therapeutic biomolecules [37]. In this context, various therapeutic applications have recently been described in regenerative medicine, showing great potential in neurodegenerative disease treatment [38].

One of the most-studied salivary exosomal biomarkers for mTBI and PCS is tau protein, a microtubule-associated protein stabilizing neuronal axons. Tau protein is also implicated in microtubule-mediated axonal transport, making it a key player in neuronal development [39]. However, the active implication of tau aggregation in predisposing one to tauopathies was previously demonstrated only for moderate to severe TBIs [40]. Despite this, it was shown that increased levels of tau protein within biological fluids are mainly present within the first 24 h post-TBI [41]. The balance between tau and its active phosphorylated form is a known biomarker for acute and chronic TBIs [42]. In several of the studies discussed herein, increased levels of tau protein originating from exosomes were found in the saliva of individuals with mTBI and PCS, indicating axonal damage and neuronal degeneration [19,25,26,27]. Additionally, exosomal phosphorylated tau was found to be elevated in the saliva of individuals with repeated mTBIs, suggesting a potential link between repetitive mTBIs and chronic neurodegenerative disorders, such as CTE. As tau protein levels increase in biological sources obtained from patients with Alzheimer’s disease (as repeatedly reported [43,44]), older studies report unclear correlations between mTBI and Alzheimer’s disease. However, more recent studies showed that mTBI could predispose one to AD and related dementias, regardless of age or sex [45,46].

Exosomal miRNAs are another class of biomolecules that have shown promise as salivary biomarkers for mTBI and PCS, as several studies discussed herein found dysregulated levels of exosomal miRNAs in the saliva of individuals with mTBI and PCS [22,27]. MiRNAs are short RNA molecules consisting of non-coding sequences that regulate gene expression. Some of the miRNAs that were isolated from post-mTBI patients were associated with dendritic differentiation and synaptic function [32]. One study suggested that dysregulated exosomal miRNAs could be associated with inflammation and neuronal repair pathways in individuals with repeated mTBI [22]. Exosomal miRNAs were also associated with neurological disease, developmental injury and abnormalities, and neuropsychiatric disease, as well as with chronic mTBI [27]. While several miRNAs were shown to be implicated in mTBI and PCS, as changes in biological fluids levels were previously documented, they were also described as potential therapeutic targets in both animal and patient studies [32].

Cytokines, such as IL-6 and IL-10, have also been investigated as potential salivary exosomal biomarkers for mTBI and PCS, and exosomal TNF-α, IL-6, and IL-10 levels were observed to be significantly increased in individuals with mTBI, as compared to healthy controls [23,24,26]. The roles of cytokines are mainly tied to the inflammatory response; however, in the brain, the activity of cytokines was also described as modulatory in pathways such as learning and memory, neuronal development and differentiation, synaptic plasticity, the blood–brain barrier, and sleep regulation [47]. Recent studies have investigated the role of salivary exosomal PRPc, XIIIa, synaptogyrin-3, IL-6, and aquaporins in mTBI and PCS and reported that the prolonged increased levels of aquaporins and IL-6 in neuron-derived exosomes might contribute to the persistent central nervous system oedema and inflammation observed in CTE [24]. In this context, the molecular dysregulations caused by mTBI target brain circulation and blood coagulation, brain water balance and edema formation, the tau-accumulation-associated signaling pathway, and the acute-phase inflammatory response. On the other hand, counteracting measures have been described to be designed for homeostasis and repair as well as the replacement of the damaged cells [48,49]. A recent animal model study showed that Il-6 and TGF-β are implicated in macrophage infiltration and subsequent tissue repair [50]. In this context, it would be interesting to study the potential of exosomal cytokines in regeneration after an mTBI event. Thus, future research could focus on describing the changes in exosomal miRNAs’ expression in correlation with mTBI-affected brain molecular pathways to further uncover the altered signaling pathways that lead to mTBI and PCS symptoms and outcomes, as well as possible means of overcoming them and preventing long-term effects.

In conclusion, using exosomal biomarkers in saliva has shown great promise in diagnosing and managing mTBI and PCS. There are several studies identifying several exosomal biomarkers for mTBI and PCS, namely, tau protein, p-tau protein, miRNAs, IL-6, and IL-10. These salivary biomarkers could provide a non-invasive diagnostic tool and assist in identifying individuals at risk of developing chronic symptoms and progression to CTE. Further research could focus on exosomal biomarkers, as they may provide valuable insight into the underlying pathophysiology of mTBI and PCS that could lead to targeted therapies. Furthermore, future studies are needed to validate these biomarkers in larger cohorts, determine their sensitivity and specificity in diagnosing mTBI and PCS, and aid in the discovery of reliable ways of separating salivary exosomes and quantifying exosomal content. Overall, exosomal biomarkers in saliva show great potential in the diagnosis and mana