From positron emission tomography to cell analysis of the 18-kDa Translocator Protein in mild traumatic brain injury

Traumatic brain injury (TBI) leads to a deleterious neuroinflammation, originating from microglial activation. Monitoring microglial activation is an indispensable step to develop therapeutic strategies for TBI. In this study, we evaluated the use of the 18-kDa translocator protein (TSPO) in positron emission tomography (PET) and cellular analysis to monitor microglial activation in a mild TBI mouse model. TBI was induced on male Swiss mice. PET imaging analysis with [18F]FEPPA, a TSPO radiotracer, was performed at 1, 3 and 7 days post-TBI and flow cytometry analysis on brain at 1 and 3 days post-TBI. PET analysis showed no difference in TSPO expression between non-operated, sham-operated and TBI mice. Flow cytometry analysis demonstrated an increase in TSPO expression in ipsilateral brain 3 days post-TBI, especially in microglia, macrophages, lymphocytes and neutrophils. Moreover, microglia represent only 58.3% of TSPO+ cells in the brain. Our results raise the question of the use of TSPO radiotracer to monitor microglial activation after TBI. More broadly, flow cytometry results point the lack of specificity of TSPO for microglia and imply that microglia contribute to the overall increase in TSPO in the brain after TBI, but is not its only contributor.

Feasibility of TSPO-Specific Positron Emission Tomography Radiotracer for Evaluating Paracetamol-Induced Liver Injury

Macrophages are activated during the early phase of paracetamol-induced liver injury (PLI). [18F]GE180 is a radiolabeled ligand that recognizes the macrophage translocator protein (TSPO). In this study, we evaluated the feasibility of a TSPO-specific radiotracer in a rat model of PLI. A rat model of liver injury was induced by intraperitoneal administration of paracetamol. [18F]GE180 positron emission tomography (PET) images were obtained after 24 h. The maximal and mean standardized uptake values (SUVmax and SUVav) of the liver and serum biomarker levels were examined. The TSPO expression level was examined using real-time polymerase chain reaction and Western blot analysis. [18F]GE180 hepatic uptake in the PLI group was significantly higher than that in the control group (SUVmax p = 0.001; SUVav p = 0.005). Both mRNA and protein TSPO expression levels were higher in the PLI group. The mRNA expression level of TSPO was significantly correlated with [18F]GE180 hepatic uptake in both groups (SUVmax p = 0.019; SUVav p = 0.007). [18F]GE180 hepatic uptake in the PLI group showed a significant positive correlation with ALT24 and ALT48 (ALT24 p = 0.016; ALT48 p = 0.002). [18F]GE180 enabled visualization of PLI through TSPO overexpression. Our results support the potential utility of hepatic uptake by TSPO-PET as a non-invasive imaging biomarker for the early phase of PLI.

Neuroinflammation and Its Association with Cognition, Neuronal Markers and Peripheral Inflammation after Chemotherapy for Breast Cancer

To uncover mechanisms underlying chemotherapy-induced cognitive impairment in breast cancer, we studied new biomarkers of neuroinflammation and neuronal survival. This cohort study included 74 women (47 ± 10 years) from 22 October 2017 until 20 August 2020. Nineteen chemotherapy-treated and 18 chemotherapy-naïve patients with breast cancer were assessed one month after the completion of surgery and/or chemotherapy, and 37 healthy controls were included. Assessments included neuropsychological testing, questionnaires, blood sampling for 17 inflammatory and two neuronal survival markers (neurofilament light-chain (NfL), and brain-derived neurotrophic factor (BDNF) and PET-MR neuroimaging. To investigate neuroinflammation, translocator protein (TSPO) [18F]DPA714-PET-MR was acquired for 15 participants per group, and evaluated by volume of distribution normalized to the cerebellum. Chemotherapy-treated patients showed higher TSPO expression, indicative for neuroinflammation, in the occipital and parietal lobe when compared to healthy controls or chemotherapy-naïve patients. After partial-volume correction, differences with healthy controls persisted (pFWE < 0.05). Additionally, compared to healthy- or chemotherapy-naïve controls, cognitive impairment (17–22%) and altered levels in blood markers (F ≥ 3.7, p ≤ 0.031) were found in chemotherapy-treated patients. NfL, an axonal damage marker, was particularly sensitive in differentiating groups (F = 105, p = 4.2 × 10 −21), with levels 20-fold higher in chemotherapy-treated patients. Lastly, in chemotherapy-treated patients alone, higher local TSPO expression was associated with worse cognitive performance, higher blood levels of BDNF/NfL, and decreased fiber cross-section in the corpus callosum (pFWE < 0.05). These findings suggest that increased neuroinflammation is associated with chemotherapy-related cognitive impairment in breast cancer. Additionally, NfL could be a useful biomarker to assess neurotoxic effects of anticancer chemotherapies.

Modelling [18F]LW223 PET data using simplified imaging protocols for quantification of TSPO expression in the rat heart and brain

Purpose To provide a comprehensive assessment of the novel 18 kDa translocator protein (TSPO) radiotracer, [18F]LW223, kinetics in the heart and brain when using a simplified imaging approach. Methods Naive adult rats and rats with surgically induced permanent coronary artery ligation received a bolus intravenous injection of [18F]LW223 followed by 120 min PET scanning with arterial blood sampling throughout. Kinetic modelling of PET data was applied to estimated rate constants, total volume of distribution (VT) and binding potential transfer corrected (BPTC) using arterial or image-derived input function (IDIF). Quantitative bias of simplified protocols using IDIF versus arterial input function (AIF) and stability of kinetic parameters for PET imaging data of different length (40–120 min) were estimated. Results PET outcome measures estimated using IDIF significantly correlated with those derived with invasive AIF, albeit with an inherent systematic bias. Truncation of the dynamic PET scan duration to less than 100 min reduced the stability of the kinetic modelling outputs. Quantification of [18F]LW223 uptake kinetics in the brain and heart required the use of different outcome measures, with BPTC more stable in the heart and VT more stable in the brain. Conclusion Modelling of [18F]LW223 PET showed the use of simplified IDIF is acceptable in the rat and the minimum scan duration for quantification of TSPO expression in rats using kinetic modelling with this radiotracer is 100 min. Carefully assessing kinetic outcome measures when conducting a systems level as oppose to single-organ centric analyses is crucial. This should be taken into account when assessing the emerging role of the TSPO heart-brain axis in the field of PET imaging.

Supervised clustering for TSPO PET imaging

Purpose This technical note seeks to act as a practical guide for implementing a supervised clustering algorithm (SVCA) reference region approach and to explain the main strengths and limitations of the technique in the context of 18-kilodalton translocator protein (TSPO) positron emission tomography (PET) studies in experimental medicine. Background TSPO PET is the most widely used imaging technique for studying neuroinflammation in vivo in humans. Quantifying neuroinflammation with PET can be a challenging and invasive procedure, especially in frail patients, because it often requires blood sampling from an arterial catheter. A widely used alternative to arterial sampling is SVCA, which identifies the voxels with minimal specific binding in the PET images, thus extracting a pseudo-reference region for non-invasive quantification. Unlike other reference region approaches, SVCA does not require specification of an anatomical reference region a priori, which alleviates the limitation of TSPO contamination in anatomically-defined reference regions in individuals with underlying inflammatory processes. Furthermore, SVCA can be applied to any TSPO PET tracer across different neurological and neuropsychiatric conditions, providing noninvasivequantification of TSPO expression. Methods We provide an overview of the development of SVCA as well as step-by-step instructions for implementing SVCA with suggestions for specific settings. We review the literature on SVCAapplications using first- and second- generation TSPO PET tracers and discuss potential clinically relevant limitations and applications. Conclusions The correct implementation of SVCA can provide robust and reproducible estimates of brain TSPO expression. This review encourages the standardisation of SVCA methodology in TSPO PET analysis, ultimately aiming to improve replicability and comparability across study sites.

De novo Neurosteroidogenesis in Human Microglia: Involvement of the 18 kDa Translocator Protein

Neuroactive steroids are potent modulators of microglial functions and are capable of counteracting their excessive reactivity. This action has mainly been ascribed to neuroactive steroids released from other sources, as microglia have been defined unable to produce neurosteroids de novo. Unexpectedly, immortalized murine microglia recently exhibited this de novo biosynthesis; herein, de novo neurosteroidogenesis was characterized in immortalized human microglia. The results demonstrated that C20 and HMC3 microglial cells constitutively express members of the neurosteroidogenesis multiprotein machinery—in particular, the transduceosome members StAR and TSPO, and the enzyme CYP11A1. Moreover, both cell lines produce pregnenolone and transcriptionally express the enzymes involved in neurosteroidogenesis. The high TSPO expression levels observed in microglia prompted us to assess its role in de novo neurosteroidogenesis. TSPO siRNA and TSPO synthetic ligand treatments were used to reduce and prompt TSPO function, respectively. The TSPO expression downregulation compromised the de novo neurosteroidogenesis and led to an increase in StAR expression, probably as a compensatory mechanism. The pharmacological TSPO stimulation the de novo neurosteroidogenesis improved in turn the neurosteroid-mediated release of Brain-Derived Neurotrophic Factor. In conclusion, these results demonstrated that de novo neurosteroidogenesis occurs in human microglia, unravelling a new mechanism potentially useful for future therapeutic purposes.

GMP-compliant fully automated radiosynthesis of [18F]FEPPA for PET/MRI imaging of regional brain TSPO expression

Background Expression of translocator protein (TSPO) on the outer mitochondrial membrane of activated microglia is strongly associated with neuroinflammation. The second-generation PET ligand [18F]FEPPA specifically binds TSPO to enable in vivo visualization and quantification of neuroinflammation. We optimized a fully automated radiosynthesis method and evaluated the utility of [18F]FEPPA, the second-generation PET ligand specifically binds TSPO, in a mouse model of systemic LPS challenge to detect TSPO-associated signals of central and peripheral inflammation. In vivo dynamic PET/MR imaging was performed in LPS-induced and control mice after [18F]FEPPA administration. The relationship between the [18F]FEPPA signal and the dose of LPS was assessed. The cytokine levels (i.e., TNF-α, Il-1β, Il-6) in LPS-induced mice were measured by RT-PCR. Standard uptake value (SUV), total volume of distribution (VT) and area under the curve (AUC) were determined based on the metabolite-uncorrected plasma input function. Western blotting and immunostaining were used to measure TSPO expression in the brain. Results The fully automated [18F]FEPPA radiosynthesis produced an uncorrected radiochemical yield of 30 ± 2% within 80 min, with a radiochemical purity greater than 99% and specific activity of 148.9‒216.8 GBq/µmol. Significant differences were observed in the brain after [18F]FEPPA administration: SUV, VT and AUC were 1.61 ± 0.1, 1.25 ± 0.12 and 1.58 ± 0.09-fold higher in LPS-injected mice than controls. TNF-α, Il-1β and Il-6 mRNA levels were also elevated in the brains of LPS-injected mice. Western blotting revealed TSPO (p < 0.05) and Iba-1 (p < 0.01) were upregulated in the brain after LPS administration. In LPS-injected mice, TSPO immunoactivity colocalized with Iba-1 in the cerebrum and TSPO was significantly overexpressed in the hippocampus and cerebellum. The peripheral organs (heart, lung) of LPS-injected mice had higher [18F]FEPPA signal-to-noise ratios than control mice. Conclusions Based on the current data on ligand specificity and selectivity in central tissues using 7 T PET/MR imaging, we demonstrate that [18F]FEPPA accumulations significant increased in the specific brain regions of systemic LPS-induced neuroinflammation (5 mg/kg). Future investigations are needed to determine the sensitivity of [18F]FEPPA as a biomarker of neuroinflammation as well as the correlation between the PET signal intensity and the expression levels of TSPO.

Detection of Alzheimer’s disease-related neuroinflammation by a PET ligand selective for glial versus vascular translocator protein

A substantial and constitutive expression of translocator protein (TSPO) in cerebral blood vessels hampers the sensitive detection of neuroinflammation characterized by greatly induced TSPO expression in activated glia. Here, we conducted in vivo positron emission tomography (PET) and in vitro autoradiographic imaging of normal and TSPO-deficient mouse brains to compare the binding properties of 18F-FEBMP, a relatively novel TSPO radioligand developed for human studies based on its insensitivity to a common polymorphism, with 11C-PK11195, as well as other commonly used TSPO radioligands including 11C-PBR28, 11C-Ac5216 and 18F-FEDAA1106. TSPO in cerebral vessels of normal mice was found to provide a major binding site for 11C-PK11195, 11C-PBR28 and 18F-FEDAA1106, in contrast to no overt specific binding of 18F-FEBMP and 11C-Ac5216 to this vascular component. In addition, 18F-FEBMP yielded PET images of microglial TSPO with a higher contrast than 11C-PK11195 in a tau transgenic mouse modeling Alzheimer’s disease (AD) and allied neurodegenerative tauopathies. Moreover, TSPO expression examined by immunoblotting was significantly increased in AD brains compared with healthy controls, and was well correlated with the autoradiographic binding of 18F-FEBMP but not 11C-PK11195. Our findings support the potential advantage of comparatively glial TSPO-selective radioligands such as 18F-FEBMP for PET imaging of inflammatory glial cells.