Brain Structural and Functional Abnormalities Associated with Acute Post-Traumatic Headache: Iron Deposition and Functional Connectivity

Background The purpose of this study was to interrogate brain iron accumulation in participants with acute post-traumatic headache (PTH) due to mild traumatic brain injury (mTBI), and to determine if functional connectivity is affected in areas with iron accumulation. We aimed to examine the correlations between iron accumulation and headache frequency, post-concussion symptom severity, number of mTBIs and time since most recent TBI. Methods Sixty participants with acute PTH and 60 age-matched healthy controls (HC) underwent 3T magnetic resonance imaging including quantitative T2* maps and resting-state functional connectivity imaging. Between group T2* differences were determined using T-tests (p < 0.005, cluster size threshold of 10 voxels). For regions with T2* differences, two analyses were conducted. First, the correlations with clinical variables including headache frequency, number of lifetime mTBIs, time since most recent mTBI, and Sport Concussion Assessment Tool (SCAT) symptom severity scale scores were investigated using linear regression. Second, the functional connectivity of these regions with the rest of the brain was examined (significance of p < 0.05 with family wise error correction for multiple comparisons). Results The acute PTH group consisted of 60 participants (22 male, 38 female) with average age of 42 ± 14 years. The HC group consisted of 60 age-matched controls (17 male, 43 female, average age of 42 ± 13). PTH participants had lower T2* values compared to HC in the left posterior cingulate and the bilateral cuneus. Stronger functional connectivity was observed between bilateral cuneus and right cerebellar areas in PTH compared to HC. Within the PTH group, linear regression showed negative associations of T2* and SCAT symptom severity score in the left posterior cingulate (p = 0.05) and with headache frequency in the left cuneus (p = 0.04). Conclusions Iron accumulation in posterior cingulate and cuneus was observed in those with acute PTH relative to HC; stronger functional connectivity was detected between the bilateral cuneus and the right cerebellum. The correlations of decreased T2* (suggesting higher iron content) with headache frequency and post mTBI symptom severity suggest that the iron accumulation that results from mTBI might reflect the severity of underlying mTBI pathophysiology and associate with post-mTBI symptom severity including PTH.

Two ve-minute runs of resting state blood oxygen level dependent (BOLD) imaging with TE of 27 ms, TR of 2500 ms, FOV 64x64x64 mm 3 , FA of 90 degrees, and voxel size 4x4x4 mm were collected while participants relaxed with their eyes closed.For each participant, the T 1 and T 2 imaging sequences were reviewed by a neuroradiologist, and imaging was excluded from further analyses if abnormal brain imaging ndings were present.

Image Postprocessing
Data pre-processing was done using SPM12 software ( Functional images were motion corrected, realigned to the rst image of the set, coregistered to the anatomical T 1 -weighted image, realigned to the average MNI template, and smoothed using an 8 mm FWHM Gaussian kernel using SPM12.Further post processing in AFNI 3dTproject included band pass ltering (0.01-0.1 Hz) after removal of nuisance signals from framewise displacement, cerebrospinal uid signal, and linear drift.The rst ve frames were excluded to allow the signal to reach steady state.
Frames with excess of 2 mm motion were also removed from the analysis.Region of interest to brain correlation maps were calculated using MATLAB.Regions of signi cant T 2 * difference between PTH and HC were used to select the regions of interest for the functional connectivity analysis.Left and right hemisphere clusters were interrogated separately.The Pearson correlation coe cients were calculated between the region of interest and the rest of the brain using in-house software written in MATLAB.The correlation maps were Fisher transformed to Z-scores for group comparisons.The symptoms included in the ICHD-3 diagnostic criteria for primary headaches were used to classify the PTH phenotypes: 35 participants with PTH had migraine-like headache phenotypes, 8 had probable migraine-like phenotypes, 14 had tension-type-like headache phenotypes, 1 had probable tension type-like headache phenotype, and 2 participants' PTH phenotypes were not classi able.

Statistical Analysis
Forty-ve percent of all PTH participants suffered a mTBI due to a fall, 30% due to motor vehicle accident, 12% due to a ght, 7% hit against object, 5% due to a sport injury, and 2% hit by object.Forty-six percent of PTH participants had one lifetime mTBI, 17% had two mTBIs, 16% had three, 5% had four, and 16% had 5 or more lifetime TBIs.For all participants, the number of reported days between mTBI and imaging ranged from 4 to 50 days with an average of 25 days (SD = 15 days).Twenty-seven participants had PTH onset immediately following injury, 23 had PTH starting within one day of the mTBI, and ten had PTH onset of > 1 day post mTBI but prior to 7 days.PTH participants had headaches on an average of 81% of the days since their TBI (SD = 28% with a range from 7-100%).Fifty-ve percent of PTH participants had continuous headaches.
the bilateral cuneus to the cerebellum.We speculate that altered structure of the cuneus and its connectivity with the cerebellum could be associated with abnormal eye movements that often occur following mTBI, such as abnormalities in saccades, smooth pursuit, and vergence [24][25][26].Unfortunately, we did not measure oculomotor dysfunction in our study.
Prior studies have demonstrated atypical structure and connectivity of DMN regions amongst those with mTBI and amongst those with PTH [23,27,28].In our study, abnormal T 2 * was identi ed in the posterior cingulate, an important hub of the DMN.Niu et al. reported disruption in the connectivity between the DMN with the periaqueductal gray amongst those with acute PTH due to mTBI which could signify impaired pain modulation following mTBI and which was a strong predictor of PTH persistence at three months post injury [23,27].Impaired pain modulation leading to oxidative stress could be a possible mechanism resulting in increased T 2 * .
Zhou et al. showed reduced connectivity in the posterior cingulate and parietal regions and increased frontal connectivity in the medial prefrontal cortex in patients with mTBI relative to HC.In this study, we also observed opposing changes in functional connectivity with some connections being stronger and others being weaker.Zhou et al. also showed that the posterior connectivity correlated positively with neurocognitive dysfunction while the frontal connectivity was negatively correlated to post-traumatic symptoms such as symptoms of postconcussion syndrome.In this study, we did not interrogate functional connectivity correlations to headache severity, but we did show that headache severity correlated negatively with iron burden in the left posterior cingulate and with headache frequency in the left cuneus.
Iron accumulation in the two regions found in this study correlated with measures of headache frequency and post-mTBI symptom severity.We found negative associations of headache frequency with T 2 * in the left cuneus, one region previously identi ed to associate with headache frequency [19,29,30].Negative association between headache frequency and decrease of gray matter volume in the cuneus was reported by Neeb et al. [30] in migraine groups compared to HCs.This nding suggests a link between pain and brain plasticity, independent of injury.The negative associations with headache frequency suggest that there may be an accumulation of iron due to recurrent headaches.

Limitations
T 2 * decrease is assumed to relate to iron accumulation, but that decrease may be due to hemorrhage, venous blood, calci cation, or changes in tissue water concentration.Future work will evaluate these contrast contributions further using phase information from quantitative susceptibility imaging.
In this study, regions with T Wellcome Department of Cognitive Neurology, Institute of Neurology, London, UK) in conjunction with MATLAB version R2019b (Mathworks, Natick, MA, USA).T 2 * maps were smoothed with a 6 mm kernel and resampled to match the T 1 -weighted images.The T 1 -weighted images were used to normalize to Montreal Neurological Institute (MNI) space along with the co-registered T 2 * to yield 1mm isotropic resolution maps.A 4 mm dilated mask was used to remove cerebrospinal uid (CSF) contamination.

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Table 1
The average T 2 * values from regions with signi cant T 2 * differences were included in a linear model to assess association with clinical variables within the PTH group.The clinical variables examined were number of lifetime mTBIs, time since most recent mTBI, the Sport Concussion Assessment Tool (SCAT) symptom severity checklist score, and headache frequency.Headache frequency was calculated as the percentage of days with headache since mTBI.The regions with signi cant T 2 * differences between PTH and HC were used as seeds for a for full brain resting state functional connectivity correlation analysis.A full factorial analysis was conducted in SPM12 with two factors: group factor with two levels for PTH and HC and region of interest factor with the number of levels equal to the number of regions of interest used, as well as covariates to compensate for age and sex.Group differences for each region of interest were examined with a F-statistic with post hoc t-tests used to assess direction of the effect.Signi cance for group differences was set to p < 0.05 with family wise error correction for multiple comparisons.Participant demographics and post traumatic headache clinical variables and phenotypes.Time since mTBI is the number of days between the date of mild traumatic brain injury and the baseline research visit.Headache frequency is reported as the percent number of days since mTBI with headache.Time from mTBI to PTH is the number of hours between the brain injury and headache onset.Values reported as mean ± standard deviation.Abbreviations: mTBI = mild traumatic brain injury, PTH = post traumatic headache, HC = healthy controls.
T 2 * differences between 60 PTH participants and 60 age-matched HC were evaluated in MNI space using a t-test within SPM12 (https://www.l.ion.ucl.ac.uk/spm/software/spm12/) compensating for age and sex.Cluster analysis was performed in SPM 12 using uncorrected p < 0.005 and volume threshold of 10 mm 3 (10 voxels) to interrogate T 2 * differences in PTH compared to HC. Cluster labelling was assisted with automated anatomical atlas ROI_MNI_V7.Results A total of 120 participants were included in the analysis.Participant demographics, headache characteristics, and mTBI characteristics are shown in Table 1.The acute PTH group consisted of 60 participants (22 male, 38 female) with average age of 42 years (SD = 14, range from 19 to 70).The HC group consisted of 60 age-matched controls (17 male, 43 female) with an average age of 42 (SD = 13, range from 21 to 71).Age matching (PTH-HC) had an average difference of 0.1 years (SD = 2, range − 3.6 to 5.3 years).
2This study was approved by the Mayo Clinic and Phoenix VA Institutional Review Boards * decreases consistently showed cerebellar connectivity disruption yet no T 2 * differences in the cerebellum are reported.The eld of view of our T 2 * sequence consistently cropped the