Validation of [18F]LW223 for TSPO PET imaging in mice and assessment of cellular TSPO expression during neuroinflammation

Neuroinflammation is associated with a number of brain diseases, including dementias, stroke, multiple sclerosis, Parkinson’s disease, depression and many more, making it a common feature of cerebral pathology. Therefore, the use of imaging techniques that are capable of detecting neuroinflammation, such as Positron Emission Tomography (PET), become valuable and versatile tools for diagnosis and monitoring of progression of these diseases. Among the best-known biomarkers for neuroinflammation in PET research is the 18kDa translocator protein (TSPO). During neuroinflammation, TSPO expression in the brain is typically upregulated, and the increase in TSPO can be detected using TSPO-specific PET radiotracers. This PhD project focused on the validation of a novel TSPO PET radiotracer, [18F]LW223, in mice and its application to image sex-dependent TSPO expression in the healthy brain, as well as in neuroinflammation resulting from bacterial endotoxin-induced systemic inflammation (lipopolysaccharide (LPS) challenge) and vascular dementia (bilateral carotid artery stenosis, or BCAS). In addition, this project aimed to investigate cellular TSPO distribution in health and under inflammatory conditions. It was hypothesised, that 1) the binding of [¹⁸F]LW223 in vivo mirrors TSPO distribution in the mouse brain, while having low non-displaceable binding, high target occupancy and affinity for the target; 2) regional [¹⁸F]LW223 binding in the murine brain differs between healthy males and females; 3) TSPO expression in the brain of mice post-systemic LPS challenge and with BCAS can be detected using [¹⁸F]LW223; and 4) in healthy brain, TSPO is mostly expressed by endothelial cells, while under neuroinflammatory conditions, its expression shifts primarily to microglia. The validation of [18F]LW223 PET imaging for preclinical use was performed in healthy young C57Bl/6J mice. Mice co-injected with [18F]LW223 and varying concentrations of unlabelled LW223 were used to estimate volume of non-displaceable binding, target occupancy and affinity of this PET radiotracer. In addition to dynamic PET imaging over 2 hours, arterial input functions (AIFs) were generated using an automated blood sampling system. In order to determine parent fraction (i.e. unmetabolised fraction) of the radiotracer in mouse blood and organs, a separate cohort of mice were injected with [18F]LW223, the tissues were collected at 2, 5, 10, 30, 60 and 120 minutes post-injection, processed and analysed using radio-high performance liquid chromatography (HPLC). To find the most suitable quantification method for [18F]LW223 PET data, multiple kinetic modelling methods with AIF, image-derived input functions (IDIFs) and population-derived input functions with metabolite correction were investigated, as well as simplified quantification methods, such as standard uptake values (SUV) and apparent volume of distribution (VTapp). In order to assess differences between male and female TSPO expression, male and female C57Bl/6J mice were subjected to 2 hours of dynamic PET imaging immediately post-[18F]LW223 injection. Volume of interest-based SUV analysis and voxel-based parametric mapping were used to determine sex-dependent TSPO expression in mouse brain. The in vivo results were compared with in vitro [18F]LW223 autoradiography and TSPO immunohistochemistry. Additionally, the distribution of TSPO in glial and endothelial cells of male and female mouse brain was assessed by double immunofluorescence staining using antibodies against TSPO and astrocytes (GFAP), microglia/macrophages (Iba1), oligodendrocytes (Olig2) and endothelial cells (CD31). After validation of [18F]LW223 in healthy animals, the radiotracer was used to image neuroinflammation resulting from acute (LPS) and chronic (BCAS) harmful stimuli. In the LPS model, mice were injected (i.p.) with vehicle, 0.5 mg/kg or 1 mg/kg of LPS from E. coli, and 24 hours later underwent [18F]LW223 PET imaging. In the LPS study, brain tissue was harvested following PET imaging session and divided into two hemispheres – one was lysed and used to measure levels of IL-1β, IL-6, TNF-α and TSPO using ELISA kits, and another was cryosectioned for in vitro [18F]LW223 autoradiography and ex vivo validation of PET results, as well as for double immunofluorescence staining with antibodies against TSPO and GFAP, Iba1, Olig2 and CD31 to assess cellular dynamics of TSPO expression under inflammatory conditions. The BCAS mixed coil model was generated by Prof Horsburgh’s group. Immediately post BCAS surgery, mice were placed on either control diet or diet containing colony stimulating factor 1 receptor (CSF1R) inhibitor GW2580 for 5 weeks, followed by PET imaging with [18F]LW223. Additionally, in order to explore translational potential of [18F]LW223, in vitro autoradiography was performed using human brain sections of healthy controls and small vessel disease (SVD) patients. The validation study showed that binding of [18F]LW223 matched the known distribution of TSPO in the mouse body, had low volume non-displaceable binding in the brain (VND) (