How does chronic neuroinflammation affect resting state functional connectivity?
Esteban Adrian Oyarzabal1,2,3, SungHo Lee1,4,5, Manasmita Das1,2, Sheng Song6,7, and Yen-Yu Ian Shih1,2

1Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 2Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 3Neurobiology Laboratory, National Institute of Environmental Health Sciences, RTP, NC, United States, 4University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 5Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, 6Neurobiology Laboratory, National Institute of Environmental Health Sciences, 7National Institute of Environmental Health Sciences, RTP, NC, United States


Chronic neuroinflammation, present in most neuropathologies, has long-term consequences on neurocircuit connectivity synchrony and strength. By implementing multi-modal techniques to quantify neuroinflammation, we found a strong association linking the intensity of neuroinflammation with depressed functional connectivity. We partly attribute these changes to neurodegeneration and the loss of central NE.


Neuroinflammation serves as a defensive response to isolate, repair or destroy injurious brain tissue that has been damaged by pathogens, senile plaques, blunt trauma, ischemia or oxidant stress.1 When neuroinflammation is persistent, such as in Alzheimer's disease, Parkinson's disease, Huntington's disease, and Multiple Sclerosis, it contributes to the degeneration of susceptible bystander neurons from microglia-mediated collateral oxidant damage.2 Though non-immunologically activated microglia are involved in plasticity to improve functional connectivity3, almost nothing is known about how their immunological activation during chronic neuroinflammation alters neurocircuit functions. Most importantly, deciphering how chronic neuroinflammation alters fcMRI is clinically relevant for early diagnosis of neurodegenerative disorders since patients present chronic neuroinflammation nearly a decade before the onset of their disorder-related symptoms. To investigate this, mice were systemically exposed to the bacterial endotoxin lipopolysaccharide (LPS) to generate a global, chronic neuroinflammation4,5 detected 12 months after onset using the inflammatory markers [18F]-PBR111 and CD11b+ (Fig 1a). This model afflicts motor balance and coordination (Fig 1b) and recapitulates the progressive pathological axis of many neurodegenerative disorders with high neuronal losses of noradrenergic Locus Coeruleus (NE-LC) and dopaminergic Substantia Nigra pars compacta (DA-SNpc) neurons and lesser losses to hippocampal and motor cortex neurons while sparing the striatum and VTA (Fig 1c). Since heterogeneous intensities in neuroinflammation are present in this model and they seem to align with regions of degeneration, we hypothesize that chronic neuroinflammation affects functional neurocircuit synchrony and strength in an intensity-depended manner. Using a multi-modal approach combining in vivo PET and fcMRI we interrogated how spatial gradations in chronic neuroinflammation intensities throughout the brain directly alter the strength of functional connectivity synchrony and neuronal metabolism in resting state neural circuits.


C57BL6/J mice were exposed to saline (n=12) or 5 mg/kg of LPS (i.p, E. coli strain O111:B4, Sigma-Aldrich L3012; n=12). Global chronic neuroinflammation was confirmed in vivo on a GE eXplore Vista PET/CT using by [18F]-PBR1116 and [18F]-FDG-PET was also performed as a brain activation surrogate. PET radiotracers (~10 MBq, i.v.) were administered 45 min prior to imaging and mice were scanned under 2% isoflurane for a 20 min PET static emission acquisition correcting for decay, attenuation, scatter and random coincidences and a 6 min CT acquisition for skull stripping. Brain images were aligned to custom templates and standardized uptake values (MBq/cm3) were calculated and transformed into normally distributed Z-scores for each function connectivity circuit volume (listed on Fig 2e) in PMOD. fcMRI were collected on a Bruker 9.4-Tesla/30-cm scanner with a BGA9-S gradient insert and imaged using a Bruker mouse receiver surface coil coupled with a transmitter volume coil. Imaging sedation of 1-1.5% isoflurane and subject physiological parameters were maintained while acquiring single-shot gradient echo EPIs in two, 10 minute sessions with bandwidth = 250 kHz, TR/TE = 3000/10 ms, labeling duration = 3.0 s with no post-labeling delay, matrix = 64x64, FOV = 1.92 cm2, slice number = 26 and slice thickness = 0.3 mm. BOLD acquisitions were slice-timing and motion corrected, aligned, spatial smoothed (FWHM = 0.6 mm), bandpass filtered (0.01-0.1 Hz) and normalized using FSL and warped with ANTs to a custom C57BL/6J probabilistic EPI atlas. Correlation maps were generated using Pearson correlations undergoing a Fisher's r-to-Z transformation through JMP (v.13, SAS) and visualized as connectivity matrices. Voxelwise functional connectivity maps were generated using AFNI and Z-scores for circuit strengths were calculated for each function connectivity circuit volume and compared to PET data.


Brain regions with greater chronic neuroinflammation intensities (Fig 2b) had suppressed neuronal metabolism as measured by PET-FDG (Fig 2c, e), reduced voxelwise connectivity strengths in function connectivity circuit volumes (Fig 2d, e) and decreased resting-state circuit synchrony (Fig 3a). Notably, chronic neuroinflammation significantly reduced connectivity strength (p<0.05) in all five major resting-state network compared to controls. Previous findings7 lead us to interrogate whether NE loss from LC degeneration could alter fcMRI connectivity during chronic neuroinflammation by exposing mice to atipamezole (Fig 3b, 1 mg/kg, s.c.) to evoke fcMRI changes through a 30% increase in central NE levels8 and found mice with chronic neuroinflammation had poor responses in fcMRI networks synchrony compared to age-matched controls (Fig 3).


Though inflammation can suppress functional connectivity in the hedonic reward circuits of patients with depression9, our study is the first to directly correlate depressions in both synchrony and circuit strengths in all major resting-state networks directly to gradations in the intensity of chronic neuroinflammation intensity. Furthermore, we suspect these changes to be a result of significant neurodegeneration and the loss of cortical NE.


We thank members of the Shih and Hong laboratories for their valuable input discussing the experimental design and results in this study. We also thank Jon Frank and Joseph Merrill of the University of North Carolina at Chapel Hill’s Biomedical Research Imaging Core for performing the PET imaging studies. E.A.O. was supported by the National Heart, Lung, and Blood Institute (NHLBI) Training Grant. M.D. was supported by the Human Frontier Science Program-Cross Disciplinary Fellowship. S.S. and J.S.H. were supported my Intramural Research Division of the National Institute of Environmental Health Sciences (NIEHS). Y.Y.I.S. was supported by the NINDS (NS091236), the National Institute of Mental Health (MH106939), the National Institute on Alcohol Abuse and Alcoholism (AA020023), the National Institute of Health UL1TR001111 sub-awards 550KR81420 and 550KR91413, the Brain and Behavior Foundation Young Investigator Award and Ellen Schapiro & Gerald Axelbaum Investigator fund, the American Heart Association Scientist Development Award (15SDG23260025), and the Department of Neurology and the Biomedical Research Imaging Center at UNC Chapel Hill.


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Figure 1. Systemic exposure to the bacterial endotoxin LPS generates a chronic, global neuroinflammation that alters motor behavior and produces discrete neurodegeneration throughout the brain. (a) Autoradiography of mice exposed to LPS 10 months prior showed that the radioligand [18F]-PBR111 corresponds to heightened densities of CD11b+ immunologically activated microglia detected by immunohistochemistry. (b) LPS mice displayed motor deficit behaviors and had (c) significant loss of noradrenergic neurons of the Locus Coeruleus and dopaminergic neurons of the Substantia nigra with lesser, yet significant, losses to neurons in the Hippocampus and Motor Cortex.

Figure 2. Chronic neuroinflammation proportionally suppresses neuronal activity and fcMRI circuit synchrony strength in mice. (a) Anatomical reference images show (b) LPS generates heterogeneous chronic neuroinflammation intensities throughout the brain as detected in vivo with PET-PBR uptake and their (c) neuronal activity as detected in vivo with PET-FDG is depressed compared to shams. (d) Connectivity strength, as derived from voxelwise correlation maps of fcMRI data, show chronic neuroinflammation suppresses connectivity strength in cortical, midbrain and limbic structures. (e) Multi-modal Z-score comparisons along functional connectivity circuit volumes confirm chronic neuroinflammation reduces network strength and neuronal activity in an intensity-dependent manner.

Figure 3. Chronic neuroinflammation alters the ability to evoke fcMRI circuit synchrony suppression by stimulating the release of the neuromodulator NE. (a) BOLD resting state fcMRI circuits are depressed in mice with chronic neuroinflammation compared to age-matched control mice. (b) Evoking the autoregulatory release of cortical NE using 1mg/kg of atipamezole generates fcMRI circuit depressions in the synchrony of all resting state networks, but hardly alters fcMRI circuits in mice with chronic neuroinflammation.

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)