Seventy-seven patients (45 male, ages of 34.7 ± 12.2 years, education level of 8.6 ± 3.8 years) with mTBI and forty-two matched healthy controls (HC, 21 male, ages of 35.3 ± 11.2 years, education level of 10.5 ± 5.2 years) were recruited in the study (Clinical trial: NCT02868684). All consecutive patients from the local emergency department (ED) with non-contrast head CT due to acute head trauma enrolled as the initial population. Inclusion criteria for mild TBI were based on the World Health Organization’s Collaborating Centre for Neurotrauma Task Force (21). Mild TBI patients were excluded:1) history of a previous brain injury, preexisting headache, neurological disease, long-standing psychiatric condition, or concurrent substance or alcohol abuse, 2) structural abnormality on conventional neuroimaging (CT and MRI), 3) intubation and/or presence of a skull fracture and administration of sedatives, 4) the manifestation of mild TBI due to medications by other injuries (e.g., systemic injuries, facial injuries, or spinal cord injury), 5) other problems (e.g., psychological trauma, language barrier, or coexisting medical conditions), 6) caused by penetrating craniocerebral injury. Patients with structural abnormality on conventional neuroimaging and a premorbid condition, such as history of a previous brain injury, preexisting headache, neurological disease, concurrent substance or alcohol abuse were excluded. MRI scanning for mTBI patients was originally evaluated within 7 days post-injury (acute phase) and follow-up at three month post-injury (chronic phase). Measures for patients were circulating markers of inflammation, clinical and neuropsychological assessments within 48 hours of MRI scans.
MTBI patients were divided into two groups based on the presence of PTH at the acute stage (within 7 days post-injury): mTBI with and without acute posttraumatic headache (APTH) according to the Third Edition of the International Classification of Headache Disorders (ICH-D-3) (22). If the headache persists for longer than three months after head trauma, it is described as the chronic/persistent PTH (CPTH/PPTH).
Healthy subjects carefully screened for history of acute/chronic pain, neurological or psychiatric disorder, were also recruited. Forty-two age-, sex- and education-matched healthy volunteers completed an identical neuroimaging scan and assessments at a single time point as a control group. The study was approved by the local ethics committee in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. (see online supplementary eMethods).
Serum biomarker collection and assay
Serum samples for both patients and controls were collected in the morning around 7-8 am. Sample were aliquoted and stored at -80℃ until the time of assay after collection and centrifugation. Serum cytokine levels (pg/mL) were measured using reagents on a Luminex multiplex bead system (Luminex Austin, TX, USA). A fluorescence detection laser optic system was used to analyze binding of each individual protein on the microsphere simultaneously, which permits multiplexed analysis of several analytes in one sample. Intra- and inter-assay coefficients of variation observed for Luminex quantification were less than 20 percent and 25 percent, respectively. Samples with levels that were undetectable by the assay were set to the value of 0.01 pg/mL. The criteria for cytokines selection were mainly based on whether it’s related to TBI or clinical symptoms such as PCS and pain function.(23, 24) The cytokines included (i) the archetypal pro-inflammatory cytokines: IL-1β, IL-6 and IL-12, and the anti-inflammatory cytokines IL-4, IL-10; (ii) chemokine (C-C motif) ligand 2 or monocyte chemoattractant protein-1(CCL2 or MCP-1) and member of the CXC chemokine family (CXCL8) IL-8; (iii) interferon-γ (IFN-γ); and (iv) tumor necrosis factor α (TNF-α).
Clinical evaluations and Pain symptom measurement
Clinical evaluations included post-injury days, duration for both the posttraumatic amnesia (PTA) and loss of consciousness (LOC). Intensity of pain symptoms were assessed by the Visual Analogue Scale (VAS, range 0-10) (25). The Pain VAS confines to patient-report (PR) measures including pain subscales for the current headache and current general pain as well as mean, best and worst levels of general pain intensity experienced in the preceding week. The VAS ranges from 0 to 10, with 0 meaning “no pain at all” and 10 “the worst possible pain” and reliably tested in previous reports(26, 27). The impact of headache on patients’ lives was also evaluated with the Short-Form Headache Impact Test (HIT-6) (28, 29).
A comprehensive neuropsychological assessments included: i) Trail-Making Test Part A and Digit Symbol coding score from the Wechsler Adult Intelligence Scale III (WAIS-III) to examine cognitive information processing speed; ii) Forward Digit Span and Backward Digit Span from the WAIS-III to assess immediate auditory span, working memory, and executive function; iii) Verbal Fluency Test to assess verbal fluency including language ability, semantic memory and executive function; iv) Depression severity was assessed using the Beck Depression Inventory (BDI-II); v) Posttraumatic stress disorder (PTSD) Checklist - Civilian Version (PCL-C); vi) Fatigue Severity Scale, Insomnia Severity Index. In addition, post concussive symptoms (PCS) were measured with the Rivermead Post-Concussion Symptom Questionnaire (RPQ)(30) consisting of 16 items, which was specifically developed to assess the severity of symptoms experienced after brain injury.
The protocol for scanning included a non-contrast CT scan for acute head injury. MRI scanning was conducted on 3T MRI scanner (GE 750) and included the T1-weighted 3D BRAVO sequence, conventional T1- and T2-weighted image, and susceptibility weighted imaging (SWI) (see online supplementary eMethods).
MRI data processing
The T1-MRI images were processed using the Computational Anatomy Toolbox (CAT12) in Statistical Parametric Mapping 12 (SPM12; https://www.fil.ion.ucl.ac.uk/ spm/software/spm12/). Anatomical images were firstly segmented into the gray matter, white matter, and cerebrospinal fluid (CSF), spatially normalized into the Montreal Neurological institute (MNI) template space, and then smoothed with an isotropic Gaussian kernel of 8 mm full width at half maximum. Group differences on GMV using the total intracranial volume, age, and sex as covariants were performed with a cluster forming (voxel-wise) threshold of uncorrected P < 0.001 and then corrected for multiple comparisons at the cluster level (p < 0.05, family-wise error (FWE) rate correction). Absolute threshold masking was set at 0.1 to avoid edge effects between gray and white matter. Region of interest (ROI) analysis was also conducted within the pain matrix including the anterior and posterior cingulate cortex, prefrontal cortex, insula, hippocampus, middle and inferior temporal gyrus, and thalamus.
To examine whether regional GMV could mediate the effect of inflammation cytokines on pain symptom in the acute PTH, a mediation analysis was performed by using the PROCESS tool(31) as implemented in SPSS v.21. Firstly, a stepwise regression model was used to calculate the odd ratio of the serum biomarker for pain symptom measurements in mTBI patients. Secondly, based on the results of the stepwise regression analysis, resulting serum biomarker was entered as the independent variable, pain symptom measurements as the dependent variable. The mean GM volume for each region showing significant group difference was tested separately as the mediator variable in the mediation analyses, and age, sex, education and injury time as covariates of no interest .
Longitudinal analysis in CPTH
Changes of the acute serum biomarker related to the pain symptom were compared over time from acute to 3 months post-injury in both mTBI + CPTH and mTBI – CPTH groups, using the general linear model of repeated measure analysis of variance (RM-ANOVA).
Statistical analysis was performed using the Statistical Package SPSS version 20. The Shapiro-Wilk W test was used to test for normality distribution of all continuous variables. Logarithmic transformations were computed if those variables that were not normally distributed (ie, the inflammatory biomarkers). The independent two-sample t-test and the Mann-Whitney test were used to compare group differences based on data normality. Chi-square analyses were applied to assess categorical variables. Continuous variables were compared between three groups using one-way analysis of variance (ANOVA), Bonferroni’s post hoc test and Kruskal-Wallis H Tests. P < 0.05 were considered to indicate a significant difference. Between-group difference in both acute and chronic post-injury GM volume abnormalities was conducted using the general linear model in the SPM12. Using a conjunction analysis,(32) the different subgroups of mTBI patients (with PTH and without PTH) were compared to each other and to controls in both acute and chronic phases respectively. Additionally, the relationship between the GM volume for each identified ROI and pain measures were conducted using the multivariate linear regression analysis after adjusting for confounding covariates (age, sex, education and injury time). For each model, pain symptom measurements (P-VAS and HIT-6) were entered as dependent variables and brain regions showing significant GMV group difference were entered as independent variables. This procedure was repeated for both initial and follow-up data, and corrected for multiple comparisons using Bonferroni correction.