Objectives: Secondary immune injury following neurotrauma is characterized by CNS-infiltration of peripheral myeloid cells (neutrophils and monocytes/macrophages) and subsequent microglial activation (1) (Supl. Fig1). Recent work has identified inflammatory injury as a key driver of functional impairment after TBI in mice (2,3). The location and temporal dynamics of myeloid cells have the potential to serve as a clinically meaningful biomarker of secondary injury after TBI (2-4). TSPO-PET is a widely used approach for assessing neuroinflammation and glial responses (5-9). However, it cannot discriminate between resident microglia and infiltrating peripheral myeloid cells. PET imaging of triggering receptor expressed on myeloid cells-1 (TREM1) was previously identified as a highly specific tool to track CNS-infiltrating peripheral myeloid cells (5-9). Here, we propose to utilize TSPO-PET and TREM1-PET to characterize the spatiotemporal dynamics of central and peripheral maladaptive myeloid inflammation after TBI in mice. Furthermore, we performed T2-weighted magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) to provide additional information to assess for non-hemorrhagic axonal injury and edema post-TBI.

Methods: Radiochemistry: Anti-TREM1-mAb was conjugated with DOTA and radiolabeled with ⁶⁴Cu. TSPO radioligand ([¹⁸F]DPA-714) was synthesized as previously described (10). TBI: Mild TBI was induced in anesthetized male C57BL/6 mice using a closed head weight drop model or sham injury (3). MRI: At 9 days post-injury (DPI) MR imaging was performed using T2 and diffusion weighted sequences (n=6 per group) (11, 12). The genu and splenium of the corpus callosum were chosen as a priori regions or interest (ROI) due to their known susceptibility for axonal injuries. A frontal cortical ROI encompassing the motor cortex was also selected. (Supl. Fig2a,b) PET: TSPO-PET was performed at 9 DPI (n=10 per group). TREM1-PET was performed in a subgroup of the same cohort of mice at 10 DPI (n=5 per group). This time-point was chosen to maximize the specificity for infiltrating monocytes and activated microglia (1). ROI were defined by semi-automated fitting of skull CT to a 3D brain atlas (Fig1a). Following TREM1-PET, mice were sacrificed, and tissues harvested for biodistribution (BioD) analysis.

Results: MRI at 9 DPI demonstrated significantly elevated T2 signal intensity in the splenium (Supl. Fig2c, p = 0.0161) and cortex (p = 0.002) but not genu of TBI compared to sham mice. Analysis of fractional anisotropy (FA) data revealed a trend towards decreased cortical FA of TBI versus sham mice (Supl. Fig2d, p = 0.0667), suggesting changes in the cortical microstructure. At 9 DPI, whole brain TSPO-PET signal was significantly increased in TBI compared to sham mice (Fig1b, p=0.002). Cortical TSPO-PET signal trended higher but was not significant (p=0.08). At 10 DPI, significantly increased TREM1-PET signal was identified in both the whole brain and cortex of TBI mice (Fig1c, p=0.03-0.04). Ex vivo BioD demonstrated significantly increased [⁶⁴Cu]TREM1-mAb signal in the bone marrow of TBI mice (vs. shams) indicating expansion of peripheral proinflammatory myeloid cells following injury (Fig1d). TREM1 spleen signal was not significantly altered at this time point. 

Conclusion:  The present work describes the potential of TREM1-PET to detect both brain and whole-body innate inflammation after TBI in mice. Significantly increased cortical T2 hyperintensity in injured mice on MRI (favored to represent edema/inflammation) corresponds with significantly elevated cortical TREM1-PET signal in the same animals. Differing cortical PET signal between TREM1 and TSPO probes suggests that these tracers are detecting separate aspects of the inflammatory response. Additional PET experiments to further characterize the spatiotemporal dynamics of myeloid inflammation after TBI at earlier time-points are ongoing. Future experiments will utilize MR-driven ROIs to better understand regional injury and relationship between MR and PET neuroinflammatory signals.

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Figure 1: CT registered to mouse 3C brain atlas for PET quantification (A). TSPO-PET 9 DPI (B) and TREM1-PET 10 DPI (C). [64Cu]TREM1-mAB BioD in bone marrow and spleen 10 DPI (D). Unpaired t-tests (*p≤0.05, ** p≤0.01).

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