Resting state fMRI signal complexity metrics indicate cerebellar cholinergic system damage in Gulf War Illness
Kaundinya Gopinath1, Bruce Crosson1, Unal Sakoglu2, and Robert Haley3
1Emory University, Atlanta, GA, United States, 2University of Houston Clear-Lake, Houston, TX, United States, 3UT Southwestern Medical Center, Dallas, TX, United States


Around 200,000 veterans of the 1991 Gulf War (GW) suffer from GW illness (GWI), which is characterized by deficits in cognitive, emotion, perception and nociception domains. Previous studies have associated GWI with exposure to neurotoxic chemicals which impair the cholinergic system. Recently, an fMRI time-series signal complexity metric, multi-scale entropy (MSE) has been proposed as a potential biomarker of abnormal neural activity in brain disorders. In this study, we examined 23 GWI patients and 30 age-matched controls with resting state fMRI. GWI veterans exhibited abnormally increased MSE all across cerebellum, implicating cholinergic damage of cerebellum as a mechanism underlying GWI.


Around 200,000 veterans (up to 32% of those deployed) of the 1991 Gulf War (GW) suffer from GW illness (GWI). GWI is a poorly understood chronic medical condition, characterized by multiple symptoms indicative of brain function deficits in cognitive, affective, perception and nociception domains 1-6. Epidemiologic and animal studies have associated GWI with exposure to neurotoxic chemicals such as nerve agents, organophosphate pesticides and pyridostigmine bromide, all of which are cholinergic stimulants that inhibit acetylcholinesterase 1,7,8. This increases acetylcholine concentration in the synaptic cleft; resulting in hyper-excitation of cholinergic synapses, which can cause persistent brain changes up to and including neuronal death 1,7-9. A number of brain regions including the cerebrum, striatum and cerebellum are richly innervated by cholinergic projections 10-12. A gap in the current state of knowledge is how cholinergic system impairment results in brain function deficits observed in GWI, since the effects of exposure to cholinergic stimulants can be heterogeneous across the brain 7,9. Recently, an fMRI time-series signal complexity metric, multi-scale entropy (MSE) has been proposed as a potential biomarker of neural activity and excitation/inhibition balance 13-15. In this study, we employed resting state fMRI (rsfMRI) to explore impairments in brain function networks in GWI with MSE.


23 GWI veterans (mean age 49.4 yrs.) and thirty healthy veteran controls (VC) (mean age 49.8 yrs.), were scanned in a Siemens 3T MRI scanner using a 12-channel Rx head coil. Written informed consent was obtained from all participants in the protocol approved by the local Institutional Review Board. RsMRI data were acquired with a 10-min whole-brain gradient echo EPI (TR/TE/FA = 2000/24ms/90°, resolution = 3mm x 3mm x 3.5mm). RsfMRI preprocessing steps included attenuation of signal related to subject-motion and physiological responses, using the AROMA technique 16, and spatial smoothing with FWHM = 6mm isotropic Gaussian kernel. Voxelwise sample entropy (SE) maps were evaluated at 10 temporal scales using the 3dMSE program in AFNI 17. The window-length (m) for MSE calculations was set to 2. In order to find the optimal distance threshold (r), MSE was evaluated at m=2, and r = 0.1 x SD to 1.5 x SD, in steps of 0.1 x SD (where SD was the standard deviation of the rsfMRI time-series) for all the VC rsfMRI datasets. Voxel MSEs were averaged across all grey matter voxels of all VCs. The plot of AUC of the resultant average MSE as a function of r, exhibited a maximum at r = 0.7 x SD, which was set as the optimal distance threshold for MSE calculations for all subjects. Between-group differences in complexity were obtained with separate 2-sample t-tests for SE at each scale. The resultant t-statistic maps were clustered and the inferences were corrected for multiple comparisons (mcc) through Monte-Carlo simulations explicitly accounting for the spatial correlation of second-level analysis residuals 18.


GWI veterans exhibited (Figure 1) significantly (mcc p < 0.05) increased SE at Scale 1 compared to VC across widespread regions in the cerebellum. MSE differences between the two groups in cerebrum and other sub-cortical regions did not achieve significance (at mcc p < 0.05). Thus, cerebellum seems to exhibit the most impairment in terms of hyper-excitation of cholinergic synapses in GWI. Cerebellum works in concert with basal ganglia, thalamus and cortex in the execution of almost all brain functions 19,20. Cerebellum projects to motor, somatosensory and auditory cortices 19-22, and cholinergic impairments indicated by these results may be the source of perceptual, perceptuomotor, and vestibular deficits, as well as confusion ataxia seen in GWI 2,8. Cerebellum is also known to take part in language 23 and other cognitive functions 23-26, which is consistent with deficits in these functions seen in GWI. Cholinergic impairment of cerebellum can also give rise to chronic pain symptoms 27, which are prevalent in GWI 4,8,28,29. These results are also consistent with structural and functional abnormalities in cerebellum observed by other groups 28,29.


The results of this study implicate hyper-excitation of cholinergic synapses in cerebellum as a putative mechanism for GWI. Future work will extend this analysis to a larger GWI patient cohort, and also examine relationship between MSE and measures of neurotoxic exposure, as well as the neuroprotective PON1 gene expression in GWI.


This work was supported by the Office of Assistant Secretary of Defense for Health Affairs, through the Gulf War Illness Research Program under Award No.W81XWH-16-1-0744. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.


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Figure 1: GWI vs VC group t-test map on fMRI signal sample entropy at Scale 1. Maps have been thresholded at mcc p < 0.05. Slice locations are provided in MNI co-ordinates.

Proc. Intl. Soc. Mag. Reson. Med. 28 (2020)