Anna Rita Daniela Coda

In vivo imaging and characterization of [18F]DPA-714, a potential new TSPO ligand, in mouse brain and peripheral tissues using small-animal PET

INTRODUCTION The translocator protein 18 kDa (TSPO), a biochemical marker of neuroinflammation, is highly expressed in the brain activated microglia and it is also expressed by peripheral inflammatory cells and normal peripheral tissues. Thus, development of radioligands for the TSPO may contribute to further understanding the in vivo TSPO function in central and peripheral inflammatory processes and other pathologies. Here, we report the biodistribution, the specific binding and the radiometabolites of [(18)F]DPA-714, a promising fluorinated PET radiotracer, in normal mice using a microPET/CT scanner. METHODS The in vivo biodistribution and kinetics of [(18)F]DPA-714 were measured in mice brain and peripheral tissues. Specific binding to TSPO sites was assessed using pharmacological competitive studies by means of saturation experiments performed by i.v. injection of 1mg/kg of unlabeled DPA-714 or 3mg/kg of unlabeled PK11195. A region of interest analysis was performed to generate time-activity curves in the brain, heart, lung, kidney, spleen and liver. Metabolites assay was performed in the plasma and peripheral organs by radio-HPLC. RESULTS [(18)F]DPA-714 reached high concentration in lung, heart, kidney and spleen, tissues well known to be rich in TSPO sites. [(18)F]DPA-714 kinetics were faster in the lung and slower in the kidney. Pre-injection of unlabeled DPA-714 or PK11195 inhibited about 80% of [(18)F]DPA-714 uptake in the lung and heart (p<0.0005). The percentage of inhibition in the kidney was lower and achieved at later times only with DPA-714 (p<0.05) but not with PK11195. Sixty minutes after radiotracer injection only unmetabolized radioligand was found in the brain, lung, heart and spleen. CONCLUSION These results suggest that [(18)F]DPA-714 is a suitable PET ligand for imaging in mice brain and peripheral tissues since it binds with high specificity TSPO binding sites and it is almost unchanged at 60 minutes after radiotracer injection in the brain and TSPO-rich regions.

Supratentorial and infratentorial damage in spinocerebellar ataxia 2: a diffusion-weighted MRI study.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal‐dominant degenerative disorder that is neuropathologically characterized primarily by infratentorial damage, although less severe supratentorial involvement may contribute to the clinical manifestation. Diffusion‐weighted imaging (DWI)–Magnetic Resonance Imaging (MRI) studies of SCA2 have enabled in vivo quantification of neurodegeneration in infratentorial regions, whereas supratentorial regions have been explored less thoroughly. We measured microstructural changes in both infratentorial and supratentorial regions in 13 SCA2 patients (9 men, 4 women; mean age, 50 ± 12 years) and 15 controls (10 men, 5 women; mean age, 49 ± 14 years) using DWI‐MRI and correlated the DWI changes with disease severity and duration. Disease severity was evaluated using the International Cooperative Ataxia Rating Scale and the Inherited Ataxia Clinical Rating Scale. Cerebral diffusion trace ( D¯ ) values were generated, and regions of interest (ROIs) and voxel‐based analysis with Statistical Parametric Mapping (SPM) were used for data analysis. In SCA2 patients, ROI analysis and SPM confirmed significant increases in D¯ values in the pons, cerebellar white matter (CWM) and middle cerebellar peduncles. Moreover, SPM analysis revealed increased D¯ values in the right thalamus, bilateral temporal cortex/white matter, and motor cortex/pyramidal tract regions. Increased diffusivity in the frontal white matter (FWM) and the CWM was significantly correlated with ataxia severity. DWI‐MRI revealed that both infratentorial and supratentorial microstructural changes may characterize SCA2 patients in the course of the disease and might contribute to the severity of the symptoms.

Head and Neck Veins of the Mouse. A Magnetic Resonance, Micro Computed Tomography and High Frequency Color Doppler Ultrasound Study

To characterize the anatomy of the venous outflow of the mouse brain using different imaging techniques. Ten C57/black male mice (age range: 7-8 weeks) were imaged with high-frequency Ultrasound, Magnetic Resonance Angiography and ex-vivo Microcomputed tomography of the head and neck. Under general anesthesia, Ultrasound of neck veins was performed with a 20MHz transducer; head and neck Magnetic Resonance Angiography data were collected on 9.4T or 7T scanners, and ex-vivo Microcomputed tomography angiography was obtained by filling the vessels with a radiopaque inert silicone rubber compound. All procedures were approved by the local ethical committee. The dorsal intracranial venous system is quite similar in mice and humans. Instead, the mouse Internal Jugular Veins are tiny vessels receiving the sigmoid sinuses and tributaries from cerebellum, occipital lobe and midbrain, while the majority of the cerebral blood, i.e. from the olfactory bulbs and fronto-parietal lobes, is apparently drained through skull base connections into the External Jugular Vein. Three main intra-extracranial anastomoses, absent in humans, are: 1) the petrosquamous sinus, draining into the posterior facial vein, 2) the veins of the olfactory bulb, draining into the superficial temporal vein through a foramen of the frontal bone 3) the cavernous sinus, draining in the External Jugular Vein through a foramen of the sphenoid bone. The anatomical structure of the mouse cranial venous outflow as depicted by Ultrasound, Microcomputed tomography and Magnetic Resonance Angiography is different from humans, with multiple connections between intra- and extra- cranial veins.

High Frequency Ultrasound for In Vivo Pregnancy Diagnosis and Staging of Placental and Fetal Development in Mice

Background Ultrasound is a valuable non-invasive tool used in obstetrics and gynecology to monitor the growth and well being of the human fetus. The laboratory mouse has recently emerged as an appropriate model for fetal and perinatal studies because morphogenetic processes in mice exhibit adequate homology to those in humans, and genetic manipulations are relatively simple to perform in mice. High-frequency ultrasound (HFUS) has recently become available for small animal preclinical imaging and can be used to study pregnancy and development in the mouse. The objective of the current study was to assess the main applications of HFUS in the evaluation of fetal growth and placental function and to better understand human congenital diseases. Methodology/Principal Findings On each gestational day, at least 5 dams were monitored with HFUS; a total of ∼200 embryos were examined. Because it is not possible to measure each variable for the entire duration of the pregnancy, the parameters were divided into three groups as a function of the time at which they were measured. Univariate analysis of the relationship between each measurement and the embryonic day was performed using Spearman’s rank correlation (Rs). Continuous linear regression was adopted for multivariate analysis of significant parameters. All statistical tests were two-sided, and a p value of 0.05 was considered statistically significant. Conclusions/Significance The study describes the main applications of HFUS to assess changes in phenotypic parameters in the developing CD1 mouse embryo and fetus during pregnancy and to evaluating physiological fetal and placental growth and the development of principal organs such as the heart, kidney, liver, brain and eyes in the embryonic mouse. A database of normal structural and functional parameters of mouse development will provide a useful tool for the better understanding of morphogenetic and cardiovascular anomalies in transgenic and mutant mouse models.

Repeatability, Reproducibility and Standardisation of a Laser Doppler Imaging Technique for the Evaluation of Normal Mouse Hindlimb Perfusion

Background Preclinical perfusion studies are useful for the improvement of diagnosis and therapy in dermatologic, cardiovascular and rheumatic human diseases. The Laser Doppler Perfusion Imaging (LDPI) technique has been used to evaluate superficial alterations of the skin microcirculation in surgically induced murine hindlimb ischemia. We assessed the reproducibility and the accuracy of LDPI acquisitions and identified several critical factors that could affect LDPI measurements in mice. Methods Twenty mice were analysed. Statistical standardisation and a repeatability and reproducibility analysis were performed on mouse perfusion signals with respect to differences in body temperature, the presence or absence of hair, the type of anaesthesia used for LDPI measurements and the position of the mouse body. Results We found excellent correlations among measurements made by the same operator (i.e., repeatability) under the same experimental conditions and by two different operators (i.e., reproducibility). A Bland-Altman analysis showed the absence of bias in repeatability (p = 0.29) or reproducibility (p = 0.89). The limits of agreement for repeatability were –0.357 and –0.033, and for reproducibility, they were –0.270 and 0.238. Significant differences in perfusion values were observed in different experimental groups. Conclusions Different experimental conditions must be considered as a starting point for the evaluation of new drugs and strategic therapies.

Molecular imaging of neuroinflammation in preclinical rodent models using positron emission tomography

Neuroinflammation (NI) is an adaptive response to different noxious stimuli, involving microglia, astrocytes and peripheral immune cells. NI is a hallmark of several acute and chronic diseases of central nervous system (CNS) and contributes to both damage and repair of CNS tissue. Interventional or genetically modified rodent models mimicking human neuropathologies may provide valuable insights on basic mechanisms of NI, but also for improving the development of new diagnostic and therapeutic strategies. Preclinical positron emission tomography (PET) allows to investigate noninvasively the inflammatory response in CNS of rodent models at a molecular level, validating innovative probes for early diagnosis, and characterizing the time course of neuroinflammatory changes and their relationship with disease progression, as well as the effects of experimental treatments with high translational potential. In particular, recent efforts of preclinical PET field are intended to develop specific and selective radiotracers that target the activation of innate immune system in CNS. Here, we have reviewed the state of art for PET in relevant rodent models of acute and chronic neuropathologies associated with NI, with particular regard on imaging of activated microglia and astrocytes.

High-Frequency Ultrasound for the Study of Early Mouse Embryonic Cardiovascular System.

An accurate diagnosis of congenital heart defects during fetal development is critical for interventional planning. Mice can be used to generate animal models with heart defects, and high-frequency ultrasound (HFUS) imaging enables in utero imaging of live mouse embryos. A wide range of physiological measurements is possible using Doppler-HFUS imaging; limitations of any single measurement warrant a multiparameter approach to characterize cardiovascular function. Doppler-HFUS was used to explore the embryonic (heart, aorta) and extraembryonic (umbilical blood flow) circulatory systems to create a database in normal mouse embryos between 9.5 and 16.5 days of gestation. Multivariate analyses were performed to explore correlations between gestational age and embryo echocardiographic parameters. Heart rate and peak velocity in the aorta were positively correlated with gestational time, whereas cardiac cycle length, isovolumetric relaxation time, myocardial performance index, and arterial deceleration time of the umbilical cord were negatively correlated with it. Doppler-HFUS facilitated detailed characterization of the embryonic mouse circulation and represents a useful tool for investigation of the early mouse embryonic cardiovascular system.