Dr. Hicks is Assistant Professor of Pediatrics, Penn State College of Medicine, Hershey, PA
Recognizing the need for objective markers of brain injury, researchers have dedicated extensive efforts to developing concussion biomarkers.
Could small molecules in spit really be used to detect a brain injury? This seemingly improbable idea has gained attention thanks to recent studies utilizing a novel molecule called microRNA (miRNA). What is miRNA? What does it have to do with concussions? Why measure it in saliva?
The medical problem
Three million concussions occur in the US each year. While the small minority of concussions in professional athletes receives much attention, two-thirds of concussions occur in children and adolescents.1 Currently, the diagnosis of concussion is clinical, based on physicians’ “best guess” and subjective symptom reports from patients. There are no objective tools that provide accurate prognoses for individualized patient management.
The lack of accurate assessment tools is problematic because concussions are associated with significant morbidity, including persistent headaches, fatigue, or concentration deficits. They are also associated with missed school/work and increased health care utilization. Uncertainty surrounding the duration and severity of concussion symptoms causes significant anxiety for patients and families.2 Although most physicians feel equipped to diagnose concussions, many report that clinical tools for patient management and family education are lacking.3 The lack of clinical support tools can lead to delays in specialist referral or implementation of rehabilitation services in the subset of children who need them most.
A scientific solution
Recognizing the need for objective markers of brain injury, researchers have dedicated extensive efforts to developing concussion biomarkers. Studies harnessing neuroimaging and electrophysiology techniques have taught us a great deal about the neurobiological response to concussion, but have not resulted in a simple test for concussion diagnosis or management.4 Alternatively, approaches measuring protein biomarkers in blood have identified some molecular hallmarks of neuronal injury. However, the ability of these proteins to model brain injury and recovery is limited by their diffusion across the blood-brain-barrier, as well as the confounding influences of exercise, age, and extra-cranial pathology.5,6 A molecule specific to the neuronal injury response, small enough to cross the blood-brain barrier, and easily measured in peripheral fluids could provide valuable information to identify, characterize, and manage concussions.
MiRNAs are short, non-coding nucleic acids that regulate protein translation in response to environmental changes (such as head injury). Their abundance, stability, and essential role regulating molecular pathways make miRNAs ideal biomarker candidates. Unlike proteins, which are easily degraded by the acidic and enzymatic milieu of the oral cavity, the small size of miRNAs gives them relative stability in saliva. In healthy human subjects, salivary miRNA profiles measured with high throughput sequencing show high reproducibility across individuals.7 An investigation of miRNA content in 12 human body fluids has found the highest numbers of miRNA species in saliva, breast milk, and seminal fluid.8 Some of the most abundantly expressed miRNAs in saliva have roles in inflammation, cell growth, and apoptosis-critical molecular pathways in the response to neuronal injury.
MiRNAs can be transported through the extracellular space by exosomes and micro-vesicles, or bound to protective protein rafts. This may facilitate transport of miRNAs from the CNS into saliva, either directly through cranial nerve innervation, or indirectly across the blood-brain barrier, or the glymphatic system. This may also explain why salivary miRNA profiles show striking similarity to those found in cerebrospinal fluid (CSF).9 Unlike general inflammatory markers, miRNAs are altered in disease-specific patterns. Therefore, the miRNAs up-regulated following a deceleration injury in the brain would likely differ from the profile seen in a musculoskeletal injury or even an ischemic brain injury.
Findings from animal models and human adults indicate changes in peripheral miRNA expression following traumatic brain injury (TBI).10-12 These peripheral changes mirror miRNA patterns in the CNS. In 18 human adults with severe TBI, serum levels of miR-16, miR-92a, and miR-765 were found to be predictive of injury in the first 24 hours, compared with healthy controls.11 Multiple logistic regression analysis combining these miRNAs had 100% accuracy at differentiating control and TBI participants. Another study involving 9 adults with mild TBI and comorbid PTSD found 4 miRNAs in peripheral blood mononuclear cells that were significantly down-regulated compared with PTSD controls.12
A novel approach
We chose to investigate salivary miRNAs as biomarkers of pediatric concussion because the majority of concussions occur in children; many children prefer saliva collection over venipuncture; saliva contains high concentrations of miRNAs; and miRNAs are crucial signaling molecules in neuronal development that may enter the saliva through multiple routes. We focused on the ability of salivary miRNAs to predict the duration and character of concussion symptoms because of the clinical need for prognostic tools.
To ensure that we selected salivary miRNAs with physiologic relevance to the CNS, we began by interrogating the CSF of children with severe TBI. A case-cohort design was employed comparing longitudinal miRNA concentrations in the CSF of 7 children with severe brain injury against 3 controls undergoing rule-out-sepsis work-ups.9 The miRNAs ‘‘altered’’ in CSF were interrogated in the saliva of 60 persons with concussion (aged 7-21 years) and compared with 18 age- and sex-matched controls. This analysis revealed that over 60% of the miRNAs in CSF were also present in saliva, and 6 had parallel changes in the 2 fluids following injury (miR-182-5p, miR-221-3p, mir-26b-5p, miR-320c, miR-29c-3p, miR-30e-5p). These 6 miRNAs accurately identified children with concussion and showed strong associations with individual symptom reports.
Remarkably, salivary miRNA levels were also associated with the duration of concussion symptoms. A comparison of salivary miRNAs among children with prolonged (≥ 4 weeks; n = 30) and acute (≤ 4 weeks; n = 22) concussion symptoms revealed that concentrations of 5 miRNAs differentiated the 2 groups with over 85% accuracy (95% CI: 0.822-0.890).13 This approach was more accurate than using symptom burden on the child sports concussion assessment tool (SCAT)-3. (0.649; 95% CI: 0.388-0.887). Initial salivary concentrations of 3 miRNAs were also associated with severity of specific symptoms 4 weeks after injury, including memory difficulty, headaches, and fatigue.
Salivary miRNA represents an easily measured, physiologically relevant, and objective biomarker with clinical potential in the diagnosis and management of concussion. Certainly, additional studies validating these results in larger cohorts are needed. Such research should: explore the influence of confounding variables on salivary miRNA expression; track patterns of salivary miRNA longitudinally; and investigate salivary miRNA patterns across the lifespan. We have already begun these follow-up studies and early results are promising. For instance, among 25 male and female collegiate distance runners, concussion-related miRNAs in saliva show little change post-exercise.
Comprehensive evaluation of injury to a complex organ like the brain will likely require a multifaceted approach (similar to myocardial infarction). Thus, collaborative investigations that employ salivary miRNAs alongside neuroimaging, electrophysiology, and measurements of balance and cognition may provide powerful tools that accurately model the neurobiological response to concussion. Ideally, in-office (or sideline) collection of saliva with a simple swab technique may one day provide physicians with a point-of-care concussion tool that also offers prognostic information used to create individualized treatment plans and impart accurate anticipatory guidance.
Dr. Hicks is a co-inventor of preliminary patents for microRNA biomarkers in disorders of the central nervous system that is assigned to the SUNY Upstate and Penn State Research Foundations and licensed to Motion Intelligence, Inc. Dr. Hicks is also a consultant for Motion Intelligence, Inc. These conflicts of interest are currently managed by the Penn State College of Medicine.
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