Livia Barenghi^1*, Alberto Barenghi¹, Matteo Vidali²

¹Department of Biomedical, Surgical and Dental Sciences, University of Milan, Via della Commenda 10, 20122, Milan, Italy.

²Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Clinical Pathology Unit, Via Francesco Sforza 28. 20122 Milan, Italy.

^*Corresponding Author: Livia Barenghi, PhD, Affiliation of author: Department of Biomedical, Surgical and Dental Sciences, University of Milan, Street Via della Commenda 10

Received: 07 September 2025; Accepted: 16 September 2025; Published: 18 November 2025

Article Information

Citation: Livia Barenghi, Alberto Barenghi, Matteo Vidali. Paediatric Traumatic Brain Injury: The Evolving Role of Blood and Salivary Biomarkers. Dental Research and Oral Health. 8 (2025): 109-132.

DOI: 10.26502/droh.00100

Abstract

Keywords

Biomarker, GFAP, S100, UCHL-1, Saliva, Pediatrics, Traumatic brain injury (TBI), Guideline, Exosomes, Reference values, Cut-offs

Article Details

1. Introduction

In Italy, current national guidelines lack specific recommendations on good laboratory, clinical, and healthcare practices for the diagnosis and monitoring of traumatic brain injury (TBI), particularly with regard to the use of biological biomarkers [1].This research is based on a critical synthesis of recent systematic reviews [2-16], original studies [17-30], and commentaries [31, 32], as well as the latest guidelines [33-38] on TBI diagnosis and prognosis in the paediatric and adult populations, with a particular focus on mild TBI (mTBI), concussion [35], and the use of diagnostic biomarkers [3,4,6,10,11,15,21,23,25-30]. TBI represents an important priority in intensive care due to the associated health problems and economic costs (estimated at $400 billion annually in the USA).It is projected to become a leading cause of death and disability by 2030 [2,39],primarily resulting from acute trauma such as road accidents, falls, sports-related injuries, and violence [35]. In children, over half (55.5%) of TBI cases are related to sports or recreational activities, and 62.4% of these patients receive medical evaluation, underscoring the need for rapid and accessible diagnostic strategies in paediatric settings [40]. Globally, the incidence rate of moderate-to-severe TBI was 182.7 per 100,000 population in 2019, accounting for over half of all TBI cases, with falls and road traffic accidents as leading causes. The Italian age-standardized incidence was notably higher, at 377 (95% UI: 319-451) per 100.000 [41]. In comparison, recent data from the United States indicate that 3.0% of the population reported a TBI in the past year, including 2.2% of children aged ≤17 years (40). Epidemiological data from Huang et al. highlight age- and sex-related differences in TBI patterns, potentially relevant to diagnostic approaches [41]. The Centers for Disease Control and Prevention (CDC) report that children aged 0-17 years account for approximately 4.1% of all TBI-related deaths [35]. This is particularly relevant given that paediatric TBI (pTBI) is the leading cause of death in this age group [42]. Furthermore, pTBI has been shown to impact brain development and cognitive abilities [35,43,44].

To date, neither a single objective laboratory test [31,45] nor harmonised neuroimaging (NI) protocols [46,47] are considered a gold standard for TBI management [48]. In fact, inconsistent findings have been reported when different biomarkers are associated with specific imaging phenotypes including diffuse axonal injury, cerebral oedema, and intracranial hemorrhage. These findings suggest a low level of diagnostic specificity and may reflect the complex and heterogeneous nature of TBI, underestimated biological variability and pre-analytical and analytical issues [6,9-11,24,26-30]. Diagnosing and predicting the outcome of pTBI remains particularly challenging, especially in cases of mild TBI (mTBI) and post-concussion syndrome (PCS) [49,50]. PCS is characterized by the persistence of symptoms following mTBI [51,52]. A recent review explored the diagnostic challenges of PCS and proposed strategies to improve both research design and clinical practice, including the use of salivary biomarkers [15].In addition, several studies and reviews have investigated brain function biomarkers in both paediatric and adult populations [3,4,6,10,11,15,17,21,23,25-30,45,46,49,51,52]. Recent guidelines highlight the potential of TBI biomarkers to limit NI, particularly in the diagnostic workup of pTBI using serum and saliva samples [5,12,15,17-22,25,28,30,31,36-38,53,54]. In a recent multicentre observational study, 84.6% of children presented with mild neurotrauma, while 14.2% and 1.2% displayed moderate and severe injuries, respectively [55]. Overall, the literature suggests that the diagnostic performances of biomarkers for TBI, particularly in terms of sensitivity and specificity, varies according to the type of biological fluid used (plasma, cerebrospinal fluid (CSF) or saliva) and across populations including adults, pediatric patients and athletes [3,12, 56-70].

Many biomarkers have been proposed for evaluating TBI. These are categorized based on their characteristics as follows: brain cell proteins (mainly S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP) and ubiquitinc-terminalhydrolaseL1(UCH-L1)); stress marker (cortisol) [17]; nerve proteins (lectin-binding glycan molecules) [23]; enzymes (α-amylase, lactate dehydrogenase (LDH) [20, 71]; messengers within the immune system (cytokines); cellular integrity proteins (protein tau and amyloid β) [72]; neuropeptides (plasma calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38), vasoactive intestinal peptide (VIP), and substance P (SP)); natriuretic peptide (N-terminal pro-B-type natriuretic peptide (NT-proBNP); acute phase proteins (C-reactive protein to albumin ratio (CRP/Alb) [71]; transport proteins (hemoglobin (Hb) and serum albumin (Alb); blood clotting (PTT test) [73, 74]; functional and non-coding (nc) RNA (i.e. miRNAs, circRNAs, lncRNAs)); nano-sized, membrane-bound particles that act as messengers in cell-to-cell communication (extracellular vesicles (EV)) [5,15,22,25]; lipid and lipid transport (apoE genotype) [76, 77]; epigenetic mechanism (BDNF gene expression) [77]; and acid/base balance (pH index) [73]. Cerebrospinal fluid (CSF) is the biofluid that most accurately reflects brain functions [70], but its use is discouraged due to several limitations, including the difficulty, pain and safety concerns related to sample collection, the complexity of CSF biomarker interpretation in paediatric neurological disorders, challenges in laboratory standardization, and the limited feasibility of CSF use in large-scale or emergency settings [11,78]. As a result, blood-derived biofluids and saliva are considered more suitable matrices for paediatric biomarker analysis. The presence of brain-derived proteins in peripheral fluids (blood, saliva, urine) relies on their ability to cross the blood-brain barrier (BBB) and vascular endothelium, particularly when injured [79,80]. These proteins can subsequently be detected in gingival crevicular fluid (GCF) and saliva, through both exocrine as well as non-exocrine pathways [81].The high vascularisation of salivary glands enables the transfer of blood-derived components, including brain proteins, into saliva, an exchange largely influenced by the molecular size of these proteins [79].

Saliva is expected to play a key role in the future diagnosis of pTBI, giving several operational advantages, including easy collection, transport, and storage, and is suitable for use in emergency settings and point-of-care testing [11,12,82]. Saliva testing is non-invasive and well accepted in pediatric population, particularly when repeated sampling is needed. Several devices are available for whole saliva collection, including those specifically designed for infants. More recently, wireless biosensors for analytes like glucose and electrolytes have been integrated into saliva collection devices (e.g. glucose-tracking wireless pacifiers) [83,84].The diagnostic potential of using saliva is partly explained by the close physiological relationship between saliva production and central nervous system regulation [81].The production and composition of saliva are subject to constant and variable influences by peripheral sensory inputs and circadian rhythms. The fine regulation of secretion depends on both the parasympathetic and orthosympathetic systems. Finally, a growing body of evidences supports the idea that neurodevelopmental disorders may be linked to the oral–brain axis, partly through mechanisms mediated by the oral microbiome [81,85,86]. Consistently, proteomic data indicate that proteins associated with neurodegenerative disorders are equally abundant in saliva (n°38; 2.5%) and plasma (n°37; 2.6%) (87). However, S100B, GFAP and UCH-L1, three well established TBI biomarkers, were absent from the top 1,000 blood-derived salivary proteins identified using the manifold ranking method [88,89], suggesting limited detectability in saliva.

Our work first outlines the key features of brain biomarkers and the clinical relevance of serum biomarkers in pediatric mild TBI (pmTBI), as well as the epidemiology of this condition in adult and paediatric populations. Then, we explore eight key thematic areas:

• a) Diagnostic guidelines and the rationale for biomarkers
• b) Neuroanatomical and functional vulnerabilities in pTBI
• c) Molecular mechanisms of injury and inflammation in pTBI
• d) Saliva as an emerging matrix for TBI biomarkers
• e) Analytical, biological and clinical challenges in biomarker use
• f) Experimental biomarkers: exosomes and nc RNAs

This work aims to support the planning of well-designed clinical trials to validate current biomarker-based best practices in the paediatric population. Establishing reliable, evidence-based reference ranges and diagnostic cut-off values could reduce the reliance on NI in cases of pmTBI [15,31].

2. Materials and Methods

2.1 Focused Question

What is the diagnostic value of serum and salivary biomarkers in paediatric patients with mild traumatic brain injury (mTBI)?

2.2 Search Strategy

Given the novelty and evolving nature of the topic, we adopted a narrative review approach, with a particular focus on the translational potential of salivary biomarkers. Although narrative synthesis is less common in this domain, we considered it more appropriate than conducting an additional systematic review, given the number of recent high quality systematic and umbrella reviews already published [3-7,10-16,36,43-46,48,52,52,57-59,61,63-65,66,68,87,117]. Nevertheless, to ensure methodological rigor, we structured the research according to the PICO model (Table 1) (population, Intervention, Comparison, and Outcome) and conducted a literature search of the PubMed (MEDLINE) and Scopus databases, based on the following three aspects: population, concept, and context.

The following keywords and MeSH terms were used in various combinations with Boolean operators (OR, AND):"traumatic brain injury", "neuroimaging", "neurodegenerative diseases", "children", "adolescent", “adult”, "patient safety", "disease prediction", "biomarkers", "astrocyte injury”, "neuronal cell injury", "salivary gland tumors", "GFAP", "S100B", "UCHL1", biological functions, "serum", "saliva", "blood", "brain derived extracellular vesicles", "blood biomarkers", "saliva biomarkers", " laboratory parameters”, “diagnostic indicators", sensitivity", "specificity", “diagnostic accuracy”. "reference values", "salivary gland", “salivary flow”, salivary flux”, “pre-analytical phase”, “molecular weight”, ”half-life/kinetics, ”isoelectric point”, “salivary gland filtration”, “blood-brain barrier”, “systematic and narrative review”, “sport”, “brain anatomy”.

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2.3 Inclusion and Exclusion Criteria

The following inclusion criteria guided our analysis:(I) English language; (II) full text available; (III) published on-line firstsbetween January 2021 and June 2025; (IV) studies conducted in high- and middle-income countries (to limit salivary variability due to malnutrition); (V) studies on COVID-19 patients (when relevant to salivary biormarker analysis). References were excluded for: (I) absence of a described methodology; (II) duplications; (III) irrelevant scope; (IV) content redundancy; (V) studies without freely accessible full text; (VI) abstract-only publications; (VII) Outdated reviews when updated or more recent versions are available.

2.4 Research

This paper represents an original translational research, focusing on the evaluation of TBI biomarkers (particularly GFAP, S100B and UCH-L1) among different biological fluids ( serum, plasma, saliva). The literature search was conducted via PubMed (MEDLINE), Scopus, and Google Scholar for studies published between January 2021 to June 2025. The final search was conducted on June 30, 2025, and data extraction spanned approximately six weeks. Subsequently, bibliographic material from the papers has been used in order to find other or older appropriate sources in relation to specific topics and operative problems. Cochrane sources (reviews, protocols, clinical answers) were reviewed to identify pharmacological interventions in TBI management. Two independent, reviewers (L.B. and A.B.) screened titles and abstracts in a blinded fashion. Discrepancies were resolved by discussion or, if needed, by consultation with a third reviewer (M.V.). A total of 354 articles were initially identified. After applying inclusion and exclusion criteria, the final number on included references was 202. The heterogeneity of study designs and outcome measures prevented quantitative meta-analysis, especially for salivary biomarkers and pre- analytical variables.

Results and Discussion

TBI represents a major cause of disability and mortality in the paediatric population, with clinical presentation, injury mechanisms, and outcomes differing substantially from those observed in adults [51]. While neurological examination and neuroimaging (primarily head CT) remain the cornerstone of diagnosis, their limited specificity [90], particularly in cases of mTBI [91], often leads to overuse of imaging procedures with associated risks, especially in children. This scenario has prompted increasing interest in the use of circulating brain-derived biomarkers to improve risk stratification and guide decision-making [30,35-38,92].

Biomarkers released into blood or other bodily fluids, following brain cell injury, may offer additional diagnostic and prognostic value. However, their integration with clinical and radiological data remains an evolving field [4,39,54,61-66,71,73,78,93]. Table 2 summarizes the key characteristics of the most studied brain biomarkers in TBI, including their biological properties and limitations [4,16,30,57,62,65,79,94-105]. Recently, some reviews and papers have been published, reporting interesting findings on biomarker application from relevant paediatric case studies [3,6,9-11,24,26-30,71,78,106-111] (Table 3) [6,9-11,24,26-30,71,110-111]. Additional epidemiological and comparative data between adult and pediatric TBI are provided in Supplementary Material S1-S2.

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Table 1: Summary of the PICO framework applied.

Table 2: Main characteristics of selected biomarkers for TBI [16,30,57,62,65,79,94-105].

Table 3: Recent evidence published on pTBI using serum biomarkers [6,9-11,24,26-30,71,110-111].

Diagnostic Guidelines and the Rationale for Biomarkers

Recent international guidelines have refined the diagnostic criteria for mTBI, placing increasing emphasis on clinical evaluation and selective use of NI [2,30,32,33,35,37-39]. The World Health Organization (WHO) defines mTBI by the presence of transient neurological dysfunction and a Glasgow Coma Scale (GCS) score between 13 and 15 at least 30 minutes after trauma. Conditions such as intoxication, pre-existing neurological disorders, or co-morbid injuries must be excluded [50, 112]. The CDC recommends combining clinical signs and risk factors [35] to determine the necessity of NI, though it acknowledges the limitations of CT scans: up to 95% of children with suspected mTBI undergoing CT have no detectable intracranial injury [39]. Importantly, CT does not exclude the presence of structural brain injury, as demonstrated by MRI findings in up to 30% of children with normal CT results. Moreover, 20–40% of patients with normal CT scan may experience long-lasting post-concussive symptoms [113]. In addition, CT is often poorly accepted in paediatric populations due to the need for sedation, radiation exposure, limited accessibility in peripheral hospitals, and associated healthcare costs. The recently modified Brain Injury Guidelines (mBIG) [114, 115], while reaffirming the relevance of CT scan in pediatric TBI, advise against routine repeat imaging in low-risk patients (mBIG 1 and 2), a recommendation supported by recent studies [90, 116]. In parallel, recent evidence highlights the clinical value of measuring plasma levels of GFAP and UCH-L1 in the early management of both adult and paediatric mTBI (pmTBI). Early sampling, within 3 to 12 hours post-injury, has been associated with increased diagnostic power. Combining GFAP and UCH-L1 has been shown to improve sensitivity in detecting clinically significant TBI in children aged 16 years or younger [30]. Moreover, biomarker-based risk stratification models, including the use of S100B as a screening tool, have been proposed to reduce unnecessary CT scans and adopted by the Scandinavian guidelines; while French guidelines adopted the use of GFAP and UCH-L1 [14,36,37,96,117,118] (Table 4).

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Table 4: Main recommendations using biomarker-based risk stratification of TBI to minimize unnecessary CT scans [36,37,96].

The development of biomarker-based Clinical Decision Support Systems (CDSS) represents a significant advancement. Some models propose scoring systems based on panels including S100B, GFAP, UCH-L1, NfL, and tau, stratifying patients into low, moderate, and high risk categories. While promising, these models require validation in children under five due to physiological differences in biomarker expression. Summary of the main recommendations are provided in table 4. Other recommendations are provided from evidences by meta-analysis and modeling [14,118],and further technical details are included in table 3 and Supplementary Material S3.The integration of biomarkers into TBI classification systems remains challenging. Reference ranges must account for age, sex, and developmental stage, and must correlate with advanced NI and clinical outcomes [9,24,28,30,94,107-109]. Studies on mTBI in animal models will enhance translational research and deepen our understanding of neurodevelopmental changes in humans (Supplementary Material S4).

Moreover, TBI pathophysiology involves both primary structural damage and secondary neuroinflammatory mechanisms, each associated with distinct temporal and molecular biomarker profiles [15,35,43,61,119-123]. This complexity is further compounded by biological variability [10,26,28,29,92,95], biomarkers extracranial origin (e.g. fracture, neurodegenerative disorders) (Table 2), biological matrix (e.g. plasma, serum, CSF, saliva) [3,12,56-65,92,114], sampling time (Tables 2-4), assay differences [95, 96], and interference from autoantibodies or pharmacological agents [96,118,119] (Supplementary Material S5). Anticoagulants use usually result in a CT scan for TBI at medium and high risk; however, only the French recommendation limits CT scan in cases of medium and low levels of S100B, GFAP and UCH-L1 (Table 4).

Finally, although blood remains the preferred matrix, alternative fluids, particularly saliva, have emerged as promising in paediatrics due to their non-invasiveness and the possibility of repeated sampling. However, salivary biomarker analysis requires rigorous control of pre-analytical variables and a deep understanding of the biological mechanisms underlying secretion. Factors such as salivary flow rate, age-related variability, and swallowing dysfunction, common in children or adults with acquired brain injuries, may influence biomarker concentrations [124-126]. These aspects are further discussed in dedicated sections below.

Neuroanatomical and Functional Vulnerabilities in Paediatric TBI

The paediatric brain presents unique anatomical and physiological features that increase its vulnerability to TBI. Factors such as a larger head-to-body ratio, underdeveloped neck musculature, higher water content, and lower myelination contribute to a different biomechanical response to trauma compared to adults [127]. As a result, injury patterns differ, with children exhibiting a higher proportion of diffuse brain injuries and different lesion topographies [127].This increased prevalence of diffuse brain injuries in children is partly explained by their open basal cisterns, which allow for a different redistribution of traumatic forces [46].

Neuroimaging findings also differ significantly: while most acute lesions involve haemorrhages, injuries in children often affect extracerebral structures, including the skull and facial bones [127]. Despite clinical suspicion, CT scans are frequently negative, normal in up to 78% of paediatric cases, even when other injuries are present [55] (Supplementary material S2a). Skull fractures are reported in approximately 5% of mild and up to 50% of severe pTBI cases, and are associated with poorer clinical outcomes [128]. Beyond its traditional mechanical role in trauma, emerging evidence suggests that skull bone marrow may contribute to the neuroinflammatory response, potentially influencing secondary injury mechanisms [128] (Supplementary Materials S4).Moreover, mild TBI can disrupt age-related brain development, leading to long-term reductions in both grey and white matter volumes and associated neurocognitive impairments [129].

The brainstem, which coordinates vital functions such as respiration, blood pressure, sleep, and swallowing, is particularly susceptible to injury in children due to its anatomical orientation [61]. This is clinically relevant, as brainstem damage may influence salivary secretion and the release of brain-derived biomarkers into saliva [81]. Cranial nerves (V, VII, IX, X, XII), which regulate salivation and swallowing, are affected in pTBI (cranial nerve injury: 6.5% (CI 5.0-8.3) and 4.7% in 1-15 yrs of age, respectively) and may contribute to secondary dysfunctions [130,131].

In children, blood–brain barrier (BBB) disruption typically affects the microvascular compartment [132], influencing the passage of molecules into the bloodstream and possibly saliva. Mechanisms allowing the passage of brain-derived molecules into saliva, such as cranial nerve transport, exosomal pathways, and the glymphatic system [5,15,22,25,81,82,87,134-138], are discussed in dedicated sections below and in more detail in Supplementary Material S2. Finally, age-dependent variability in cerebral blood flow may further modulate biomarker kinetics, although its precise impact on biomarker levels remains unclear [133].

Molecular Mechanisms of Injury and Inflammation in pTBI

Paediatric TBI triggers a multifactorial cascade of molecular events that contribute to progressive cellular damage beyond the initial mechanical insult [61, 120]. These include excitotoxicity, calcium overload, oxidative stress, mitochondrial dysfunction, and neuroinflammation. Gene expression studies in animal models have revealed altered regulation of pathways involved in energy metabolism, signal transduction, cell adhesion, and transcription [4,7,120]. The characteristics of each fluid brain biomarker (Table 2) influence its ability to reflect primary versus secondary injury mechanisms. Interpretation is further complicated by pre-analytical factors (e.g., sampling time), biological variability in paediatric patients (Table 3), analytical variability, and potential extracranial confounders (Table 2) [139]. A wide range of fluid biomarkers have been proposed for mTBI, including indicators of astrocytic injury (e.g., S100B, GFAP), neuronal and axonal damage (e.g., NSE, UCH-L1, αII-spectin, tau proteins, neurofilaments), blood–brain barrier disruption (e.g., occludin) [140], and neuroinflammation (e.g., IgA, ILs, MMPs, S100A12) [141]. Neuropeptides including CGRP, PACAP-38, VIP, and substance P were significantly elevated in young children with mTBI, particularly in those with CT-positive findings, suggesting diagnostic utility [110]. Emerging markers such as exosomes and miRNAs are under investigation, but currently lack specificity regarding injury severity or anatomical localization. Genetic factors, including single nucleotide polymorphisms in APOE, BDNF, COMT, and ion channel genes (e.g., CACNA1A, ATP1A2), may contribute to individual susceptibility and outcomes in pTBI [7,142]. The APOE genotype, particularly the ε4 allele, has been associated with pTBI outcomes, although its prognostic impact appears time-dependent and may differ from that observed in adults [143,144]. While the molecular consequences of concussion and subconcussive impacts remain poorly defined, elevated levels of brain-enriched proteins such as NfL, GFAP, and autoantibodies, persisting months after injury, have been associated with long-term complications and may support extended monitoring [13]. Salivary IgA-related immune responses are also being explored as potential biomarkers of asymptomatic brain injury [141]. A machine learning-based prognostic model for pTBI has been proposed, combining clinical parameters (e.g., GCS, pupillary response, location of the head haematoma) and a risk score based on laboratory indicators (e.g., LDH, NT-proBNP, pH, Hb, Alb, CRP/albumin ratio) [71]. Although coagulopathy is a recognized complication in TBI, paediatric data on the predictive value of biomarkers, such as copeptin, S100A12, the neutrophil to lymphocytes ratio, IL-33 and galectin-3, for coagulopathy and progressive haemorrhagic injury remain limited [139,145]. Beyond structural and metabolic pathways, inflammatory mediators have received increasing attention. Cytokines such as IL-1β, IL-6, TNF-α, IL-10, and IFN-γ orchestrate both local and systemic immune responses involving resident CNS cells and circulating immune cells. In pTBI, the pro-inflammatory response appears more intense and prolonged than in adults. IL-6, in particular, has pleiotropic effects in the CNS, including modulation of acute-phase proteins, immune cell activation, blood–brain barrier permeability, and cerebral oedema through aquaporin-4 upregulation [4,146]. Recent studies have reported elevated serum levels of IL-6, IL-8, and TNF-α in pmTBI patients compared to healthy controls [27,147,148]. Extracellular vesicle–associated IL-6 was significantly increased within hours after injury in adolescent athletes [149]. Preliminary evidence also suggests that combining IL-6 with other biomarkers—such as NfL, GFAP, or NT-proBNP—may help rule out intracranial injuries, potentially reducing unnecessary CT scans and observation stays [111]. Conversely, general inflammatory indices (e.g., systemic immune-inflammation index or neutrophil-to-lymphocyte ratio) currently lack validation in this context [106,150].

Saliva as an Emerging Matrix for Tbi Biomarkers

Recent studies have explored the use of saliva as a matrix for detecting biomarkers of mTBI, both in adults and children (Table 5; Supplementary Material S6) [5,12,15,17-23,25,151-155]. In pTBI, the focus has been on brain-specific proteins such as S100B, GFAP, and UCH-L1, as well as non-specific biomarkers including Beclin1, IL-6, IL-8, D-dimer, miRNA, mitochondrial DNA, and soluble NCAM [15,92]. In adults, saliva has been investigated as a potential source for detecting cortisol, EVs, GFAP, NF-L, S100B, t-tau, UCH-L1, and CRP (Supplementary Material S6). Although these findings support the feasibility of salivary testing, the detectability of brain biomarkers in saliva varies markedly. This is partly due to differences in molecular weight, which affects their diffusion or transport from blood to saliva. For instance, GFAP and UCH-L1 show low saliva/blood ratios, whereas S100B reaches a ratio of approximately 0.8, indicating more efficient passage or different release mechanisms (Table 2). Notably, these differences are not solely attributable to molecular size, as local release from inflamed oral tissues, altered salivary flux, salivary gland AQP, and altered oral functions may also play a role as reported in details below. Approximately the point of insertion of the table 5.

Table 5. Recent evidence published on mTBI and pTBI using salivary biomarkers [5,12,15,17, 19-23,25,152-153].

Analytical, Biological and Clinical Challenges in Salivary Biomarker Use

For what has been shown so far, saliva appears to have genuine potential as a diagnostic fluid in the context of traumatic brain injury, offering a non-invasive and physiologically meaningful alternative to blood-based testing [81,82]. However, caution should be exercised when using saliva for TBI biomarker detection, as it is associated with a range of biological, pre-analytical and analytical confounders that may limit its clinical applicability (Supplementary Material S7) [79,99].

The Influence Of The Oral Health Status

Biological variability includes oral health status, age, and sex. Conditions such as gingivitis and periodontitis alter salivary concentrations of proteins such as S100 proteins, GFAP, and NSE. For example, increased levels of salivary and GFC S100A8 and S100A9 have been found in individuals with active disease, whereas these proteins were downregulated in patients with gingivitis [156]. Accordingly, the expression of GFAP and amyloid beta peptides expression (which has antimicrobial effects on oral pathogens) is increased in GCF from patients with gingivitis and periodontitis [157,158]. Moreover, increased levels of albumin and haemoglobin subunits in GCF and saliva of patients with gingivitis and periodontitis are consistent with damage to the gingival epithelial barrier, as well as the well- known presence of blood residues after tooth brushing [81,82,159-161]. NSE, being present in neurons but also in erythrocytes and platelets, lacks specificity for TBI when measured in saliva from inflamed oral environments.

Salivary cytokine concentrations, including IL-1β, IL-6, IL-8, and IL-10, have been shown to remain stable across age (4–18 years) and between sexes [162]. However, they are significantly influenced by periodontal health and salivary flow. Gingivitis, which is common during orthodontic treatment in children, leads to elevated levels of several cytokines [82]. Moreover, higher salivary flow rates are associated with lower cytokine concentrations, complicating their interpretation. A careful assessment of oral inflammatory status is therefore essential when interpreting salivary biomarkers in pTBI. Further studies are needed to better understand the possible links between oral microbial changes and cytokine levels in this context [85,86].

Regarding miRNAs, although several are promising as TBI biomarkers and appear unaffected by oral disease, their expression depends on collection timing, glandular origin, epithelial cell desquamation, and oral microbiota [163-165]. Some miRNAs associated with post-concussive symptoms overlap with those implicated in oral cancers (let 7a-3p, miRNA 133 a-5p, miR 769-5p, miR21-5), orthodontic remodeling (Let 7a-3p), or cleft lip/palate, limiting their specificity [166,167].

Salivary Flux in Paediatric and Adult Population

Salivary flow rate varies with age, sex, body weight, type of stimulation, and neurological condition [81]. High flow rates are physiological in infants and typically decline with age [81]. Excessive drooling (>3.5 mL/min) becomes pathological after the age of 4 and may occur in children with cerebral palsy, TBI, or other neurodevelopmental disorders. In healthy children, stimulated saliva shows higher flow rates than unstimulated, with older children exhibiting greater differences [162].The mean salivary flow rate was 0.8 ± 0.5 ml/min in children (7.8±2.4 years) and 1.5±0.8 ml/min in adolescents (15.1±1.7 years) [162]. The median of unstimulated salivary flow rate was 0.87 (0.54, 1.11) ml/min for boys, 0.65 (0.37, 0.98) ml/min for girls and 0.76 (0.49, 1.05) ml/min overall [168]. Obesity is associated with a modest reduction in stimulated flow, particularly during adolescence [169].

Neurological damage affects salivary gland innervation: parasympathetic input drives secretion, while sympathetic tone regulates duct contraction [170]. In this context, chronic sialorrhoea, though relatively rare, may indicate underlying neurological conditions, including TBI [171].The hypothalamic-pituitary-adrenal (HPA) axis may be altered post-TBI [4]. While data in paediatrics are limited, adult mTBI is associated with a transient increase in salivary cortisol, despite preserved circadian rhythm [103]. Long-term survivors of pTBI generally show normal or recovered HPA function. Chronic fatigue is frequently reported after TBI, but does not appear to be clearly associated with HPA axis dysfunction. In fact, morning salivary cortisone levels were higher in TBI survivors, who have a high prevalence of fatigue, compared to healthy controls.

Although rare, chronic sialorrhoea [172] presents with recognizable clinical signs, including facial skin maceration, oral infections, and respiratory or fluid balance disturbances.

Effects of Drugs

Pharmacological agents may influence biomarker detection through several mechanisms, including upregulation or suppression of protein synthesis, modulation of salivary gland function and flow, drugs [173]. Drugs like aspirin, clomipramine, curcumin, and methamphetamine affect GFAP expression; others like olopatadine, cocaine, and dopamine have been reported to effect S100B or UCH-L1 expression and then could interfere with the detection of S100B or UCH-L1 in saliva (97)(Supplementary material S5).

Pre-Analytical Factors

The timing of sample collection is one of the main confounding factors in TBI biomarker analysis. Available data indicate substantial heterogeneity: samples are collected on admission in 30.4% of cases, within 6 hours in 10.1%, within 12 hours in 4.1%, within 24 hours in 28.7%, after 24 hours in 16.6%, and are unspecified in 10.1% of cases [92]. This wide variability, especially in delayed sampling (ranging from <30 minutes to 14 days), hinders the interpretation of biomarker kinetics, particularly for miRNAs, and makes it difficult to assess the effects of sex, diet, exercise, and circadian rhythm [5]. French guidelines recommend collecting serum samples within 3 hours for S100B and GFAP, and within 3 to 12 hours for UCH-L1 (Table 4; Supplementary Material S3) [96]. Other studies, such as Manley’s, have reported meaningful levels even at 24 hours post-injury [37]. According to the Oris group, S100B, GFAP, and UCH-L1 are robust biomarkers with good pre-analytical stability in serum or plasma EDTA. S100B is stable at room temperature for up to 8 hours or refrigerated for 48 hours; GFAP and UCH-L1 remain stable for at least 3 days at 4–5°C, and for several months when frozen at -20°C or -80°C [9,62,95,96]. S100B tolerates up to five freeze–thaw cycles [174]. From an endogenous interference standpoint, S100B and UCH-L1 are unaffected by haemolysis due to their absence in erythrocytes, while GFAP may be susceptible [96]. IFUs (Instructions for Use) for tests such as Liaison® (CLIA) and Abbott’s GFAP/UCH-L1 recommend avoiding haemolysis, lipemia, buffy coat contamination, and mechanical stress (e.g., vortexing) [175,176].

For saliva, stability data are scarce [9,62,95,96]. Salivary proteins may undergo modification (e.g., extensive proteolytic cleavage, partial deglycosylation, and protein-protein complex formation) and degradation by the oral microbiome. Importantly, common sample preservatives (e.g. sodium azide) may interfere with assays [82]. In addition to stability, sample collection protocols themselves remain poorly standardised. Collection methods, such as drool, swab, or unstimulated saliva, are heterogeneous and are seldom adapted to paediatric populations [82]. Important factors like circadian rhythm, flow rate, periodontal status, and, especially, centrifugation are often neglected. These aspects are less relevant in emergency settings for moderate/severe TBI, but are critical in research and clinical use of salivary biomarkers in pmTBI.

Analytics

A wide range of analytical methods has been employed for the quantification of brain-specific biomarkers, particularly S100B, GFAP, and UCH-L1, primarily in research contexts [2-5,6,10,12,16,18-22, 31,57,82,92,152]. Available techniques for blood-based detection include ELISA (most commonly used), ECLIA, LIA, IRMA, IFMA, each with distinct analytical characteristics [9].The Oris group provided a comparative overview of commercial platforms in current use, reporting information such as manufacturer, analyser type, sample volume, reaction time, detection technique, and decision thresholds [96]. The analytical variability of GFAP and UCH-L1 assays in serum or plasma has also been documented, including the impact of autoantibodies (e.g., anti-GFAP, anti-S100B), which may lead to overestimation due to analytical interference [62,96,9,25,26]. The analytical performances of commercially available assays for S100B, GFAP, and UCH-L1 have been extensively evaluated and are generally considered acceptable for clinical use. Detailed comparative analytical data are reported in Supplementary Material S8. Although assays from different manufacturers showed strong correlation, they did not exhibit complete agreement, with systematic differences in both measured concentrations and decision thresholds. To date, all manufacturer-approved methods for these brain biomarkers are validated for use in serum or plasma, not saliva. Nonetheless, several exploratory techniques have been applied to saliva in pTBI, including immune enzymatic assays, proteomics, DNA fingerprinting, and RNA sequencing. However, the stability of biomarkers in saliva, especially when entrapped in EV, remains uncertain—as does the influence of salivary pH compared to serum (Supplementary Material S7). Currently, cortisol is the only salivary test that is CE-IVD marked and validated for clinical use. Other salivary assays—such as those for α-amylase, CRP, IL-1, IL-6, cytokine panels, secretory IgA, and markers of blood contamination like transferrin—remain confined to the research setting, with no regulatory approval for diagnostic application [82].

Additional Challenges in the Use of Saliva for Biomarker Assessment of Ptbi in Clinical Settings

Here, we aim to discuss other factors that could influence specificity. Currently, the specificity is insufficient at 45% for S100B, 11–41% for GFAP and UCH-L1, even when serum samples are used [96,177]. Beyond analytical and pre-analytical challenges (Table 2; Supplementary Material S5), the interpretation of salivary biomarkers for pTBI is further complicated by a series of additional biological and physiological factors (Table 3-5; Supplementary Material S3). These include the potential extracranial origin of brain proteins [15,31,66], embryological overlap between neural and salivary tissues, protein release and clearance mechanisms, as well as individual variables such as periodontal status, renal function, or local inflammation (Table 6) [82,86,95,96,99,122,138,174,177-195] (Supplementary Material S9). Taken together, these elements introduce substantial variability and may confound the diagnostic interpretation of salivary measurements in both research and clinical settings.

Approximately the point of insertion of the table 6.

Table 6: Additional biological and physiological factors influencing salivary biomarker interpretation [82,86,95,96,99,122,138,139,174,177-195].

Experimental biomarkers: exosomes and non-coding RNAs

Non-coding RNAs (ncRNAs), particularly miRNAs, are highly expressed in the nervous system and play key roles in neuronal physiology, including protein regulation, synapse maturation, and neural circuit formation [5,57,66,68,100,149,196-199]. Their detection within EVs — including exosomes — is being explored in TBI as a means to monitor injury mechanisms and recovery [68,100,200]. EV may include brain-specific proteins (e.g., GFAP, NF-L, UCH-L1), nucleic acids, and metabolites, potentially reflecting the cell of origin and ongoing pathological processes. However, clinical use remains experimental due to challenges in EV isolation, assay standardisation, and uncertainties about BBB passage [163,201]. In pTBI, elevated levels of exosomal GFAP and neurofilament light chains have been reported, along with marked changes in salivary EV RNA profiles [57,121]. In particular, upregulation of complement system mRNAs (e.g., C1QB, C4A, C1QA, C1S) has been observed in patients with acute post-traumatic headache, suggesting that salivary EV analysis may help monitor mTBI complications [54,202].

Conclusion

While the evidence surrounding blood and salivary biomarkers, particularly exosome miRNAs, in the diagnosis and management of pTBI is promising, current data remain insufficient to fully elucidate their roles. Saliva, especially in pediatric populations, offers distinct organizational advantages, including non-invasiveness and the potential for repeated sampling. However, further research is needed to establish standardized protocols and clarify whether salivary biomarkers simply reflect molecules already present in the blood, detect brain-derived molecules earlier than blood tests, or identify molecules that do not appear in blood at all. The biological significance of salivary biomarkers in pTBI remains unclear, and current findings do not provide a complete understanding of the extent to which salivary markers can enhance diagnostic precision. Furthermore, there is a pressing need to investigate the impact of biological, pre-analytical, and analytical variability on salivary biomarker levels, including the influence of salivary flow rate, oral health, and circadian rhythms, which may all play a role in the variability observed in both clinical and research settings. To move forward, well-designed studies are required to establish robust pediatric reference intervals and cut-off values for salivary biomarkers, which remain a key area of uncertainty. Additionally, personalized monitoring of concussion in athletes or individuals with chronic conditions should be further explored to optimize the clinical utility of these biomarkers. The development of reliable serum and salivary tests for routine clinical use is still in its early stages, and cross-platform comparisons remain challenging. Emerging technologies such as wireless biosensors and AI-generated feature clusters for diagnostic, prognostic, and therapeutic applications hold great promise but require further validation. As research progresses, it is essential that both pre-analytical and analytical variabilities are well understood and controlled to ensure that future recommendations in clinical practice are evidence-based and applicable across diverse healthcare settings.

Statements

Ethic approval

Not need

Conflict of interest

None

Author’s contribution

Conceptualization, LB, MV; data curation, LB, AB, MV; forma l analysis, LB, AB, MV; funding acquisition, LB; investigation, LB , M.V; methodology, LB, AB, M.V; project administration, LB; resources LB; supervision, MV; validation, LB, AB, MV; writing-original draft, LB; writing-review and editing, LB , M.V.

Declaration on the use of AI

The authors declare that they have not used AI-tools for writing and editing the manuscript.

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Acknowledgments

The text has been checked by a common word processor and DeepL Write for "spelling" and "grammar".

Funding

The publication costs of this work were supported by Integrated Orthodontic Services Srl, Lecco, Italy.

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