Coda, Anna Rita Daniela (2017) In vivo imaging of TSPO in mouse model of neuroinflammation using small-animal PET with [18F]DPA-714. [Tesi di dottorato]

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Introduction: Neuroinflammation (NI) may play an important role in stroke, neuroinflammatory and neurodegenerative diseases, involving microglia, astrocytes and peripheral immune cells. The translocator protein 18 kDa (TSPO) is an interesting marker of in vivo neuroinflammation because it is highly expressed by activated microglia under pathological conditions. In vivo PET imaging may provide new insight on the dynamic, topography and extent of NI since the earliest stage. Aim: To validate biodistribution and specific binding of [18F]DPA-714, a promising fluorinated radiotracer that targets the TSPO, in healthy mice and in mouse models of neurodegeneration and neuroinflammation using small-animals PET and ex-vivo methods. Methods: PET/CT was used to measure kinetics of [18F]DPA-714 in brain and peripheral tissues of normal mice, while specific binding to TSPO was evaluated using competitive studies with 1mg/kg of DPA-714 and 3mg/kg of PK11195. Metabolites assay was performed in plasma and peripheral organs by radio-HPLC. For mouse models of amyotrophic lateral sclerosis (SOD1 G93A) and of multiple sclerosis (PLP-EAE) and their control animals, in vivo [18F]DPA-714 PET/CT and ex-vivo immunofluorescence studies were performed. Results: In healthy mice [18F]DPA-714 reached high concentration in lung, heart, kidney and spleen, tissues well known to be rich in TSPO sites. Pre-injection of unlabeled DPA-714 or PK11195 inhibited about 80% of [18F]DPA-714 uptake in the lung and heart (p<0.0005). The percentage of inhibition in kidney was lower with DPA-714, but not with PK11195. Sixty minutes after radiotracer injection only unmetabolized radioligand was found in the brain, lung, heart and spleen. In the symptomatic SOD1 G93A mice, [18F]DPA-714 SUV ratio of cerebellum, brainstem and cervical spinal cord, obtained by normalizing these regions activities to that of the frontal association cortex, was increased if compared to the values in WT SOD1 mice. Immunofluorescence studies showed that TSPO expression was increased in brainstem nuclei and spinal cord of symptomatic mice and was colocalized with increased Iba1 staining. In EAE mice, the radiotracer uptake in olfactory bulbs, cerebellum and brainstem showed a significant increase in symptomatic mice, if compared to control mice, while [18F]DPA-714 uptake in the upper tract of cervical spinal cord was not significant. Immunofluorescence studies suggest that increased expression of TSPO in the posterior part of brain with cerebellum and brainstem and spinal cord was significantly expressed in Iba1 positive cells. Conclusion: Results obtained until now, support the high relevance to combine preclinical PET and ex-vivo studies for cellular characterization of the PET signal in vivo. The use of murine models of human disease may allow the evaluation of the effects of new therapies and the characterization of cellular counterparts of in vivo molecular biomarker by combining immunohistochemistry.

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