Abstract
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Objectives: Parkinson’s disease (PD) is the most prevalent movement disorder and one of the most common neurodegenerative conditions, second only to Alzheimer’s disease. The pathogenesis of PD is characterized by the loss of dopaminergic neurons in the substantia nigra and the accumulation of misfolded ⍺-synuclein protein within the cytoplasm. Importantly, PD is also strongly associated with aberrant adaptive and innate immune system responses, including microglial activation and infiltration of peripheral myeloid cells into the central nervous system (CNS). TREM1 is a promising new biomarker of proinflammatory innate immune cells including macrophages, microglia, monocytes, and neutrophils. Herein, we evaluate the utility of TREM1-PET for detecting innate immune activation in the CNS of mice after intra-striatal injection of the selective dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA) - a widely-used animal model of PD.
Methods: Twenty C57/BL6J mice were stereotaxically injected with either 6-OHDA (10 µg/µL in 1µL; n = 14) or saline (1 µL; n = 6) in their left striatum (coordinates: A/P = 0.5, L = 1.8, D/V = -3.5; Fig. 1). Additionally, three TREM1-knockout mice were injected with 6-OHDA (n = 3). On either day 7 or 14 post-surgery, mice were administered with [64Cu]TREM1-mAb (n = 18) or [64Cu]Isotype control-mAb (n = 5) and imaged by whole-body PET/CT after 19 hours. Ex vivoautoradiography (ARG) was performed to obtain high resolution images of tracer uptake within the striatum, and ratios of tracer signal in the ipsilateral versus contralateral striatum were quantified for both PET and ARG. Statistically, all groups were compared to the saline control cohort.
Results: Copper-64 radiolabeling of TREM1 mAb (chelator:mAb ratio = 0.96) and isotype control (chelator:mAb ratio = 1.9) resulted in 25% and 80% decay-corrected radiochemical yield, respectively (radiochemical purity > 99%). PET/CT images revealed TREM1-positive activated myeloid cells in the damaged brain hemisphere of 6-OHDA injected mice. PET image analysis verified significant tracer signal within the ipsilateral striatum of 6-OHDA compared to the saline-injected mice at 7 days post-surgery (P < 0.0146; Fig. 1a), which was corroborated by ex vivo ARG (P < 0.0202; Fig. 1b). Specificity of this signal was shown by the lack of significant binding in the ipsilateral striatum of TREM1-KO mice in addition to 6-OHDA mice imaged with [64Cu]Isotype control-mAb - all 7 days post-surgery. Conversely, there was no difference between saline mice and those injected with 6-OHDA at 14 days post-surgery, indicating that TREM1-PET may only be useful as an early biomarker of neuroinflammation in this model.
Conclusions: TREM1-PET represents a promising, highly specific, approach for visualizing the presence of early innate immune activation in PD rodent models. Notably, elevated levels of activated myeloid cells could be detected within the CNS of 6-OHDA injected mice in vivo using [64Cu]TREM1-mAb. Further investigation of this tracer is warranted in other PD rodent models and human postmortem tissue to assess its translational potential. Supporting Data: Figure 1. A) PET image analysis of mice injected with 6-OHDA or saline (N = 23; Saline n = 4, Day 7 n = 3, Day 14 n = 8, Isotype Ctrl n = 5, TREM1-KO n = 3), showing the ratio of tracer signal in the ipsilateral striatum over that in the contralateral striatum. B) Ex vivo autoradiography (ARG) of mice induced with 6-OHDA or saline, showing significant (P < 0.0202) tracer binding in the ipsilateral striatum over the contralateral striatum. C) Site of stereotaxic injection of 6-OHDA or saline in mouse brain - coronal view. D) Ex vivo ARG images of coronal mouse brain sections (40 μm thickness) showing tracer uptake in the striatum and injection site of 6-OHDA induced mice, compared to saline-injected mice, isotype controls, and TREM1-knockout mice. White dashed lines depict the outline of each coronal brain section.
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