Effects of soccer heading on ocular-motor function, brain-derived blood biomarker, and neuronal activity Keisuke Kawata, PhD, Assistant Professor Kinesiology, School of Public Health, Indiana University 1025 E. 7th Street, SPH C215 Bloomington, IN 47401 Hannah Block, PhD, Assistant Professor Kinesiology, School of Public Health, Indiana University Allison Gruber, PhD, Assistant Professor Kinesiology School of Public Health, Indiana University Zhongxue Chen, PhD, Associate Professor Epidemiology and Biostatistics Indiana University Sharlene Newman, PhD, Associate Professor Psychological and Brain Sciences Indiana University Table of Contents: Study Schema 1.0 Background 2.0 Rationale and Specific Aims 3.0 Inclusion/Exclusion Criteria 4.0 Enrollment/Randomization 5.0 Study Procedures 6.0 Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or Others 7.0 Study Withdrawal/Discontinuation 8.0 Statistical Considerations 9.0 Privacy/Confidentiality Issues 10.0 Follow-up and Record Retention 1.0 Background In the United States, every year approximately 1.7 million people sustain a traumatic brain injury (TBI) with the majority (80% or 1.4 millions) of these injuries falling into the category of concussion or mild TBI (mTBI).1 Concussion is classified as a mild form of traumatic brain injury, while changes in neural function following concussions are far from benign. Concussed athletes frequently experience lingering neurological symptoms, including headache, irritability, blurred vision, and confusion.2 In respect to these symptoms, researchers have utilized various metrics to study brain damage, including brain-derived blood biomarkers, sensory systems, and neuroimaging. However, due to exceedingly high intra-group variability in the concussed cohort, to date there is no gold standard metric to gauge severity of brain damage following head impacts.3 One possible explanation of the high variability is due to subconcussive head impact, defined as an impact to the head that does not cause any symptoms of concussion, regardless of magnitude of the impact. It, however, has the potential to cause insidious effects in the brain if sustained repetitively over time.4 An average athlete in contact sports experiences nearly a thousand subconcussive hits per season.5 While the vast majority of research has focused on a single impact that elicits concussion (e.g., disorientation, headache), many athletes are frequently exposed to subconcussive head impacts prior to the concussive blow.6, 7 Thus, it remains completely unknown whether neuronal damage and sensory deficit are caused by a single concussive blow, repetitive subconcussive impacts before the concussive blow, or both. Ocular-motor System Recently, the ocular-motor system has garnered significant scientific attention because of its sensitivity to various severities of brain injury.8 The ocular-motor system orchestrates accommodation and vergence, and their concomitant adjustment enables individuals to visualize an object at various distance, direction, and speed.9 Nearly 50% of the neural connections in the human brain, including cortical, cerebellar, and brainstem networks are involved in vision and eye movement functions. Since most head impacts cause the brain to bounce linearly and rotationally within the skull, almost 90% of concussed individuals exhibit some form of visual dysfunction.10, 11 For example, a notable decline in saccades –rapid changes in eye orientation– has been observed in concussed boxers, football, and rugby players.12, 13 Similarly, a near point of convergence (NPC), which is the closest point one can visualize an object before diplopia occurs, was impaired by 3-fold in concussed athletes and soldiers.14, 15 Neuroimaging confirmed that these ocular-motor impairments were in concert with altered neuronal activity after concussion.16 Previously, for the first time we have demonstrated that repetitive subconcussive head impacts could significantly impair NPC in soccer17 and football players.18 More intriguingly, the NPC has predicted concussions before they occurred. In our football study, we tracked changes in NPC in 25 players, and 3 of 25 players were diagnosed with a concussion (Fig 1: unpublished). There was a biphasic increase in NPC: 1) acute increase after 1st full-gear practice (Pads-ON1) compared with pre-practice, and 2) on the day of concussion, 3 players showed abnormally high NPC before the practice. Due to absence of concussion signs and symptoms, the players participated in the practice. Following moderate intensity of impacts, the 3 players became unconscious for < 30 seconds and diagnosed with a concussion by team athletic trainers. Despite it is preliminary, the ocular-motor function has the potential for a paradigm shift in concussion diagnosis. However, it is imperative to first understand the acute effects of subconcussive head impacts on the ocular-motor system. After Fig 1. The near point of Concussion convergence (NPC) “predicted” 22 Concussion After concussion before it occurred. 20 During Concussion Practice Three players showed near identical 18 trends. Acute NPC increase was Concussion 16 Abnormally During observed after Pads-ON1 practice High Practice (full-gear practice), then remained high 14 without normalized to baseline level, 12 indicating slow recovery nature of the 10 Abnormally ocular-motor system. Player B and C Player A High 8 Increase sustained concussions during Pads- Player B Increase On 3 practice, and Player A sustained 6 Plateau a concussion during Pads-On 4 4 Player C Increase practice. Both time, NPC before the 2 Stable practice was abnormally(cm) high (withConvergence no Point Near 0 outward symptoms), suggesting Pre Post Pre Post Pre Post Pre Post Pre Post particular vulnerability of the players baseline PadsOFF PadsON1 PadsON2 PadsON3 PadsON4 on that day. Blood Biomarker Importantly, identifying a biomarker that can detect cumulative risk for developing neurodegenerative disease is a paradigm shifting. A Particularly Interesting New Cysteine Histidine-rich (PINCH) protein is an adaptor protein involved in cell maintenance and survival of neuronal cells.19 PINCH is nearly undetectable in a healthy brain. However, PINCH expression is upregulated as neuronal axons experience a high rate of strain forces, leading to disruption of tau –an axon stabilizing protein– from axonal microtubules (Fig 2). When tau disruption occurs, PINCH binds to tau and makes it difficult to degrade and clear from the brain.20 Consequently, tau protein accumulates in the brain (Fig 2), as often observed in Alzheimer’s disease and chronic traumatic encephalopathy (CTE).21-23 If PINCH is detectable in the brain or even in the blood, neurons are mostly likely injured. Thus, this biomarker is potentially a groundbreaking because it carries an early sign of neurodegeneration. Fig 2. PINCH binds to disrupted tau and In the preliminary work, we first examined plasma PINCH in exacerbates tau aggregation. P, phosphate. Figure modified epilepsy patients, given that epilepsy is considered a form of from Kawata et al.23 neurotrauma due to neuronal hyper-excitability. We found that a robust expression of PINCH was detected in the blood of epilepsy patients while undetectable in healthy controls (Fig 3: in print).24 Similarly, 10 out of 16 collegiate football players showed noticeable increases in plasma PINCH level at post-season compared with pre-season baseline (Fig 4: unpublished). Since none of 16 athletes were diagnosed with a concussion during the season, the increased level of plasma PINCH indicates the neuronal damage from cumulative subconcussive impacts. Neuronal Activity Peripheral diagnostic tools for brain injury have an immense clinical implication, however those methods are insufficient to truly gauge neuronal dysfunction after head hits. Electroencephalography (EEG) has produced promising results in patient with Epilepsy Patients (n=7) A Healthy Control (n=5) Medically Controlled Medically Refractory 41kDa PINCH (35 -37kDa) 26kDa Fig 3. PINCHB 51kDa protein was detectable in mesial temporal epilepsy patients’ blood. Robust plasma expression of PINCH was observed in both medically controlled (no seizure) and refractory41kDa (transient seizure) epilepsy patients, while no PINCH in healthy controls. PINCH 1 2 3 4 5 6 7 8 Co n tro l A (35 -37kDa) B 26kDa * 1 2 3 4 5 6 7 8 41kDa LB OL WR OL DL LB DL DB Control PINCH (35 -37kDa) 26kDa 9 10 11 12 13 14 15 16 Co n tro l Pre-Season Post-Season * 9 10 11 12 13 14 15 16 41kDa OL RB TE LB DL OL TE WR Control PINCH (35 -37kDa) 26kDa Pre-Season Post-Season 1 2 3 4 5 6 7 8 Co n tro l A B Fig 4. Post-season plasma PINCH was notably higher than pre-season baseline. 1 2 3 4 5 6 7 8 10 out of 16 players showed increase in plasma PINCH at post-season. * Player 5 and 41kDa LB OL WR OL DL LB DL DB Control 13 did not play for the season due to surgery. Control: a subject with no history of head PINCH trauma. LB, linebacker; OL, offense lineman; WR, wide receiver; DL, defense lineman; (35 -37kDa) DB, defensive back; RB, running back; TE, tight end. 26kDa concussion, with reduced mean frequency of alpha, reduced power in the alpha and beta frequency bands,9 hypercoherence10 11 12between13 frontal14 regions,15 and 16decreasedCo n tro l gamma frequency.25-29 In addition to EEG, transcranial magnetic stimulation (TMS) has garnered Pre-Season Post-Season significant attention in scientific society recently, due to its non-invasive method to 9 10 11 12 13 14 15 16 41kDa OL RB TE