Edited by: Firas H. Kobeissy, University of Florida, USA
Reviewed by: Deborah Shear, Walter Reed Army Institute of Research, USA; Angela M. Boutte, Walter Reed Army Institute of Research, USA; Ralph George Depalma, Department of Veterans Affairs, USA
This article was submitted to Neurotrauma, a section of the journal Frontiers in Neurology.
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Although mild traumatic brain injury (mTBI), or concussion, is not typically associated with abnormalities on computed tomography (CT), it nevertheless causes persistent cognitive dysfunction for many patients. Consequently, new prognostic methods for mTBI are needed to identify at risk cases, especially at an early and potentially treatable stage. Here, we quantified plasma levels of the neurodegeneration biomarker calpain-cleaved αII-spectrin N-terminal fragment (SNTF) from 38 participants with CT-negative mTBI, orthopedic injury (OI), and normal uninjured controls (UCs) (age range 12–30 years), and compared them with findings from diffusion tensor imaging (DTI) and long-term cognitive assessment. SNTF levels were at least twice the lower limit of detection in 7 of 17 mTBI cases and in 3 of 13 OI cases, but in none of the UCs. An elevation in plasma SNTF corresponded with significant differences in fractional anisotropy and the apparent diffusion coefficient in the corpus callosum and uncinate fasciculus measured by DTI. Furthermore, increased plasma SNTF on the day of injury correlated significantly with cognitive impairment that persisted for at least 3 months, both across all study participants and also among the mTBI cases by themselves. The elevation in plasma SNTF in the subset of OI cases, accompanied by corresponding white matter and cognitive abnormalities, raises the possibility of identifying undiagnosed cases of mTBI. These data suggest that the blood level of SNTF on the day of a CT-negative mTBI may identify a subset of patients at risk of white matter damage and persistent disability. SNTF could have prognostic and diagnostic utilities in the assessment and treatment of mTBI.
Mild traumatic brain injury (mTBI), alternatively referred to as concussion, is the most common neurological injury and affects over 1.5 million children and adults each year in the United States alone, and hundreds of thousands of military personnel worldwide (
Blood-based biomarkers for brain damage have long been evaluated as potential prognostic measures in mTBI, but none have emerged thus far as a means of identifying those cases of mTBI with evolving brain damage leading to long-term dysfunction at an early and potentially treatable stage. For example, a number of proteins expressed predominantly in the nervous system become detectable in the blood during the acute post-injury period in subsets of mTBI cases. Blood levels of the astrocyte-enriched proteins S100β and glial fibrillary acidic protein (GFAP), along with the neuron-enriched neuron-specific enolase (NSE), ubiquitin C-terminal hydrolase L1 (UCH-L1), and a proteolytic fragment of tau are reportedly elevated following injuries categorized as mild based on clinical examinations using the Glasgow Coma Scale (
Given the challenge of early prognosis of functionally disruptive CT-negative mTBI, it is important to evaluate new blood-based biomarkers for neurodegeneration, and explore whether a combination of biomarkers, advanced neuroimaging, and neuropsychological methods may be more effective when used together than any single approach. Toward this end, we have discovered and begun to characterize several new candidate neurodegeneration biomarkers for acute brain damage, based on proteins that are released from degenerating neurons (
The Institutional Review Boards of the University of Pennsylvania and Texas Medical Center, Houston, reviewed and approved the study. All participants in this study provided written informed consent (or assent if written consent was given by the minor’s parent) and were recruited and assessed with approval from and according to the ethical guidelines of the Institutional Review Boards of the recruiting institutions. All procedures were conducted in accord with the ethical standards of the Helsinki Declaration of 1975, as revised in 2000 (
This study on neurodegeneration biomarkers examined 38 participants with plasma collected within 24 h of injury. Of those, 17 sustained mTBI, 13 sustained an OI, and 8 were UCs. These participants were part of a larger study comprising right-handed participants of ages 12–30 years, who were recruited and tested on neuropsychological and brain imaging measures at baseline (within 96 h of injury), and at follow-up sessions at 1 month (neuropsychological measures only) and 3 months. Participant recruitment was from a random, unselected series of patients admitted to emergency centers in the Texas Medical Center, Houston, including Ben Taub General Hospital, Texas Children’s Hospital, and Memorial Herman Hospital, or, for the UC group, from the greater Houston metropolitan area. The smaller biomarker study subgroup was selected randomly from the overall mTBI study, and did not differ significantly from the larger study sample on age, socioeconomic status (SES), race, gender, GCS score, or Extracranial Injury Severity Score (ISS).
The 17 participants providing plasma samples that experienced mTBI, as defined by criteria from the Centers for Disease Control, had an injury to the head from blunt trauma, acceleration, or deceleration forces with one or more of the following conditions: (1) observed or self-reported confusion, disorientation, or impaired consciousness, dysfunction of memory at the time of the injury, loss of consciousness lasting <30 min; and, (2) symptoms such as headache, dizziness, fatigue, irritability, and poor concentration soon after the injury. Additional inclusion criteria were a Glasgow Coma Scale score of 13–15 upon examination at an emergency center, no abnormal findings on head CT, duration of loss of consciousness for no more than 30 min, post-traumatic amnesia for <24 h, and an Abbreviated Injury Score (AIS) ≤3 and an ISS of <12 modified to exclude the head region. Comparator participants were of two cohorts. For one, participants with OI were recruited <96 h post-injury provided they met the following criteria: right-handed, 12–30 years old, no loss of consciousness, no post-traumatic amnesia, no overt intracranial injury, AIS <3 for any region of the body and an ISS ≤12, and a normal brain CT (if done). A second UC cohort consisted of eight healthy participants who had not sustained any injury, but were similar to the two injury groups in age, gender, and level of education.
Exclusions included non-fluency in either English or Spanish, failure to provide adequate contact information for scheduling follow-up assessments, blood alcohol level (200 mg/dL, previous hospitalization for head injury, pregnancy when screened prior to brain imaging, pre-existing neurologic disorder associated with cerebral dysfunction and/or cognitive deficit (e.g., cerebral palsy, mental retardation, epilepsy) or diagnosed dyslexia, pre-existing severe psychiatric disorder (e.g., bipolar disorder, schizophrenia), and contraindications to undergoing MRI. The OI comparison group was included to control for risk factors (
Participants were administered tests of cognition and assessed for symptoms related to post-concussive injury. For comparison with neurodegeneration biomarker findings, we analyzed data from three domains, speed of processing, executive memory, and cognitive flexibility, along with symptoms of concussion. The analyses were conducted by investigators blinded to the plasma biomarker data.
Rivermead Post-Concussion Symptoms Questionnaire [RPCS; (
Symbol-Digit Modalities Test [SDMT; (
KeepTrack task [KT; (
All participants underwent MRI without sedation on a Philips 3.0 T Achieva scanner. Rigorous quality assurance testing was performed including American College of Radiology phantom testing: no concerns with quality assurance were noted during the course of the study.
An axial single-shot spin-echo echo-planar imaging sequence with 30 diffusion-encoding directions was used for DTI acquisition. Other parameters included a 256 mm field of view, an acquisition voxel size of 2 mm × 2 mm × 2 mm, repetition time of 11,526 ms, echo time of 51 ms, sensitivity encoding (SENSE) reduction factor of 2, two B factors (0 s/mm2 low B and 1000 s/mm2 high B), with two acquisitions to average the signal of the two DTI scans in order to ensure better signal-to-noise ratio. DTI acquisition consisted of 70 slices. A SENSE eight-channel head coil was used.
The corpus callosum, right and left uncinate fasciculi, and right and left frontal lobes were selected as structures of interest due to their known vulnerability in DTI studies of TBI and their presumed relation to the measures of speed of cognitive processing, memory updating, and executive function, and post-concussion symptoms. Additionally, DTI measurement of these structures has been shown to be reproducible both between and within raters on quantitative tractography using previously published protocols. In this study, DTI data were analyzed twice by a single rater to establish intra-rater reliability using intra-class correlational coefficients (ICCs). A subset of the images was analyzed by two raters to establish inter-rater reliability. ICCs for all measurements were above 0.95.
The sandwich immunoassay for quantifying calpain-cleaved SNTF from human plasma is a modification of a method published previously (
Control experiments were performed to distinguish SNTF-related signals from non-specific signals emanating from heterophilic substances that are present in a subset of human plasma samples and confound attempts to measure very small amounts of target antigen (
A total of 38 randomly selected participants in an ongoing larger study of mTBI provided plasma samples on the day of injury for quantification of the neurodegeneration biomarker SNTF: 17 were diagnosed with mTBI and 13 with OI, whereas 8 were UCs. The biomarker study subgroup did not differ from the overall study group in terms of initial injury severity, age, gender, or other factors (Table
Overall group mean (±SD) ( |
Biomarker group mean (±SD) ( |
||
---|---|---|---|
Age at baseline | 20.2 (±5.4) | 20.5 (±5.8) | 0.80 |
SES | −0.0028 (±0.79) | −0.039 (±0.72) | 0.80 |
Race % non-black | 61 | 60 | 0.87 |
Gender % female | 33 | 26 | 0.38 |
GCS (mTBI)% <15 | 23 | 24 | 0.85 |
Non-cranial injury severity | 0.93 (±1.17) | 1.37 (±1.42) | 0.13 |
In comparison with the UC group, the mTBI group demonstrated overall cognitive performance deficits at 3 months post-injury on the SDMT, KT test, and the cognitive component of the RPCS, similar to reports from other studies [e.g., Ref. (
We evaluated SNTF as a candidate plasma biomarker for human mTBI. This αII-spectrin fragment is generated by the calpain family of cysteine proteases (
To examine the relationship between plasma SNTF levels on the day of mTBI and DAI, the 28 participants among the mTBI, OI, and UC cohorts with usable neuroradiological data were dichotomized as either SNTF positive or negative, and the two groups were evaluated comparatively for white matter structural abnormalities by DTI. Compared with the 19 SNTF negative cases analyzed by DTI within 4 days of injury, the 9 SNTF positive cases (7 mTBI and 2 OI) as a group exhibited significant reductions in FA and increases in ADC in the corpus callosum and uncinate fasciculus (Table
Region/metric | Mean (SD) all SNTF−( |
Mean (SD) all SNTF+ ( |
Effect size | |
---|---|---|---|---|
Corpus callosum | ||||
FA | 0.496 (0.02) | 0.479 (0.01) | 0.034 | 0.91 |
ADC | 0.821 (0.03) | 0.839 (0.02) | 0.13 | 0.63 |
Uncinate fasciculus, left | ||||
FA | 0.405 (0.02) | 0.388 (0.02) | 0.09 | 0.73 |
ADC | 0.754 (0.03) | 0.775 (0.03) | 0.14 | 0.63 |
Uncinate fasciculus, right | ||||
FA | 0.389 (0.01) | 0.367 (0.02) | 0.001 | 1.48 |
ADC | 0.774 (0.02) | 0.798 (0.03) | 0.035 | 0.89 |
Frontal lobes, left | ||||
FA | 0.394 (0.02) | 0.383 (0.02) | 0.26 | 0.47 |
ADC | 0.765 (0.02) | 0.782 (0.02) | 0.07 | 0.77 |
Frontal lobes, right | ||||
FA | 0.382 (0.03) | 0.381 (0.02) | 0.95 | 0.03 |
ADC | 0.783 (0.02) | 0.794 (0.02) | 0.15 | 0.59 |
Long-term behavioral studies have provided evidence that ∼15–30% of CT-negative patients with mTBI develop brain functional disability that can persist for many months post-injury (
Test | All SNTF+ | All SNTF− | Effect size |
---|---|---|---|
Symbol-digit modalities test, written (total correct responses) | 52.00 (12.1) | 63.47 (14.9) | 0.88 |
KeepTrack task (percent correct recalled) | 88.89 (7.8) | 92.72 (5.6) | 0.63 |
RiverMead post-concussion symptoms (total score) | 9.44 (10.89) | 6.37 (11.08) | 0.28 |
Plasma SNTF on the day of mTBI also correlated with recovery of cognitive performance. Among the 13 mTBI participants evaluated by the oral SDMT in both the acute (1–4 days) and long-term (3 months) post-injury time periods, test scores for the SNTF – cases improved by 17 points (±5.7 SEM), whereas those for the SNTF+ cases worsened by 2.6 points (±2.7). The difference in 3 months recovery of cognitive performance as a function of dichotomized plasma SNTF levels was significant (
In this study, we provide evidence that the blood level of the neurodegeneration biomarker SNTF identifies mTBI patients on the day of their injury likely to have both white matter changes with advanced neuroimaging suggestive of DAI, and also cognitive dysfunction that persists for at least 3 months. Whereas a number of brain-enriched proteins have been evaluated before as candidate prognostic markers for cases of mTBI with negligible CT findings, including the astrocyte-enriched S100β and GFAP along with the neuron-enriched NSE, cleaved tau, a C-terminal fragment of αII-spectrin termed SBDP145, and UCH-L1, none has demonstrated a prognostic relationship with structural signs for white matter injury or functional signs for impaired cognition (
αII-Spectrin N-terminal fragment is an especially plausible biomarker for DAI thought to underlie long-term brain functional impairments after mTBI (
The relationship between dichotomized plasma SNTF on the day of injury and structural differences in the corpus callosum and uncinate fasciculus in the acute post-injury period (Table
Increased plasma SNTF post-concussion is related not only to structural evidence for DAI, but also functional evidence for long-term cognitive impairment. Whereas a subset of the participants with mTBI exhibit no discernible deficits on a battery of cognitive, somatic, or emotional tests post-injury, a second group shows performance deficits that resolve over time, while a third group develops impaired brain performance persisting for at least 3 months post-injury. Strikingly, the dichotomized plasma level of SNTF measured on the day of injury is related to cognitive dysfunction at 3 months, as evidenced by a significant deficit in the SNTF positive group in the SDMT and trends toward impairments in the KT test (Table
The current study relating plasma SNTF to both structural and functional outcomes after mTBI is preliminary in nature and has several limitations, including a small sample size. For example, this study is insufficiently powered to address whether the absolute level of plasma SNTF might correlate with the severity of long-lasting behavioral dysfunction among participants functionally impacted by mTBI. The data reported here relating plasma SNTF to DTI changes in multiple axon tracts, coupled with evidence that this marker accumulates in damaged axons (
These unresolved issues take on increased urgency given the vital importance of early prognosis of functionally impactful mTBI. Despite being commonly associated with negligible head CT findings, mTBI is prevalent among both civilian and military populations and can lead to long-term brain dysfunction in as many as 15–30% of cases. A validated prognostic method for mTBI would have major utilities, including: (i) for stratifying mTBI cases and selecting a high risk population best suited for clinical trials of experimental neuroprotective treatment interventions; (ii) for serving as a surrogate endpoint for clinical neuroprotectant treatment trials; (iii) for rapidly identifying mTBI cases most likely to benefit from early initiation of rehabilitation therapies designed to improve functional outcomes; and (iv) for identifying sports and military participants at increased risk of further brain damage and disability. The current study relating SNTF to both radiological evidence for DAI and psychological evidence for long-term cognitive dysfunction raises the possibility that plasma neurodegeneration biomarkers such as SNTF may have important applications for the clinical evaluation and medical treatment of mTBI.
The research was conceived by Robert Siman, Harvey S. Levin, and Douglas H. Smith. Robert Siman and Nicholas Giovannone measured plasma levels of SNTF. Gerri Hanten conducted behavioral analyses of the study participants. Elisabeth A. Wilde, Stephen R. McCauley, and Jill V. Hunter conducted radiological analyses of the participants. Xiaoqi Li performed statistical analyses of the behavioral, radiological, and plasma biomarker data. Robert Siman, Gerri Hanten, Elisabeth A. Wilde, Stephen R. McCauley, Harvey S. Levin, and Douglas H. Smith wrote the manuscript.
A provisional patent application has been filed by the University of Pennsylvania on the use of SNTF as a prognostic biomarker for concussion, with Robert Siman named as inventor. There are no other commercial or financial relationships that could be construed as a potential conflict of interest.
This research was supported by a grant from the National Institute of Neurologic Disorders and Stroke (P01 NS-056202 to Douglas H. Smith). We thank Amanda Barnes, Trevor Wu, Ana C. Vasquez, Melisa Frisby, Greg S. Vogt, Joshua Cooper, and Claudia Robertson for their essential contributions to enrolling patients and deriving plasma samples. We gratefully acknowledge the contribution of Ponnada Narayana and Vipulkumar S. Patel in implementation and execution of the imaging sequences, and Zili D. Chu for his role in data processing. We thank Shawn Roberts and Daphne Georlette of Meso Scale Diagnostics for providing the SECTOR Imager 2400 electrochemiluminescence reader and data analysis software, along with advice on their use. We thank the participants and their families for their involvement in this research.