Development of innovative neuroimaging techniques in laboratory animals for applications in neuroscience.

Molecular imaging, which allows the real-time visualization, characterization and measurement of biological processes, is becoming increasingly used in neuroscience research. The most advanced imaging techniques such as single photon emission computed tomography (SPECT), positron emission spectroscopy (PET), computed tomography (CT), and functional magnetic resonance imaging (fMRI) have opened up the possibility of non-invasively analysing brain functions in both health and disease states. Laboratory animals, and rodents in particular, are essential in neuroscience research. By allowing selective neural and pharmacological manipulations, animal studies provide the unique opportunity to unravel the relationships between brain function and behavior. Since the neural underpinnings of complex behaviors are highly comparable in mammals of different species, the information obtained from animal research can be readily translated to humans. Furthermore, animal paradigms allow precise manipulation of experimental conditions, thus controlling for the confounding variables often present in human studies. Last, animal studies are a mandatory step to test drug efficacy in psychiatric diseases, and to determine potential side effects of drugs. Mice and rats have long served as the preferred species for biomedical research due to their anatomical, physiological, and genetic similarity to humans. Advantages of rodents as animal models include their small size, ease of maintenance, short life cycle, and abundant genetic resources. However, due to their small size, their study with PET/SPECT/CT techniques needs dedicated imaging tools with high spatial resolution, especially in the case of brain investigations. Indeed, clinical scanners used for human imaging are bulky and provide a spatial resolution of the order of tens of millimeters which is inadequate to that required for small animal studies (≤ 1mm). The general aim of my PhD project was to develop an innovative high spatial resolution SPECT system for laboratory animals, in order to overcome the limitations of current technologies in terms of spatial resolution and sensitivity, thus allowing insights into the complex functional organization of the rodent brain. Furthermore, to validate this new system and pave the way for its future applications in preclinical neuroscience research, I used this new technology to better characterize a recently developed rodent model of Autism Spectrum Disorder (ASD). ASD is one of the most severe paediatric psychiatric conditions, in terms of prevalence, outcome, impact on families and society. To date, no specific treatments for ASD are available yet. In this context, the key contribution of neuroimaging to the field is the capability to study higher neurobehavioral functions – including the activation or deactivation of given neurotransmitter systems during certain behavioural tasks – as well as the ability to investigate in vivo the biochemical changes underlying aberrant behavioural traits in longitudinal studies. In the first part of my PhD project, I studied the role of SPECT and PET small animal imaging systems for a better understanding of brain functioning in health and disease states. In particular, I evaluated the role of SPECT and PET small animal imaging systems in psychiatric disorders, focusing on the changes in metabolic and neurotransmitter activity in various brain areas during task-induced neural activation with special regard to the imaging of opioid, dopaminergic, and cannabinoid receptors. In this phase, I also carried out the initial feasibility studies of the system by simulating and designing the components of a scintigraphic detector (Chapter 3). To select the detector optimal arrangement, the entire design process has been aided by the use of Monte Carlo (MC) simulations focused to assess the detector behaviour. All the geometries and the materials have been defined according to those that would have been used in the actual physical detector. Indeed, MC simulations allow evaluating the detector behaviour and then selecting the optimal arrangement in order to achieve the desired system performance. As it turns out, brain imaging in rodent models faces a number of challenges as it acts within the boundaries of current imaging. The technical considerations about strengths and weaknesses of the different solutions, led me to develop the first prototype of a new high-performance scintigraphic detector named HiRIS2 (High- Resolution Imaging System) originally based on a position-sensitive photomultiplier tube (PSPMT) H9500 Hamamatsu coupled to a Cerium doped Lutetium-yttrium oxyorthosilicate (LYSO) pixelated scintillator crystal and a low-energy tungsten collimator with parallel square holes (Chapter 4). The results showed a detector performance suitable for application on small animals but, giving that the overall objective was to develop an improved scintigraphic system, I further made an upgrade of the system by assessing the performance of the new PSPMT Hamamatsu H13700 with 256 anodes, which replaced the Hamamatsu H9500 model. Furthermore, the LYSO scintillator was also replaced with the promising CRY018 scintillator crystal, which provides high light output, a medium density, a short decay time and excellent energy resolution, and unlike the former, has no intrinsic radioactivity. In detail, the adoption of the new PSPMT required the development of new dedicated compact electronics: a resistive chain readout was optimized and integrated with an ADC system. Moreover, the detector was engineered from both the mechanical and the electronic points of view. In fact, the entire system was miniaturized in order to reduce the overall dimensions. Finally, I started the mechanical design of the SPECT scanner, consisting of two detectors mounted in opposition on a rotary stage, allowing their rotation around the experimental subject. Then, motion software was developed: the kinematic software debugging and testing was assessed and the whole mechatronic system was implemented. The scanner prototype assessment was carried out by using several radioactive phantoms and the tomography images have been reconstructed using the Software for Tomographic Image Reconstruction (STIR) framework (Chapter 5). At this point, to enhance the imaging capabilities achievable with traditional neuroimaging systems, I implemented a method called super spatial resolution (SSR). The main aim was to demonstrate that, by combining several images containing slightly different perspectives of the same scene, it is possible to improve the overall image quality. This is achieved by moving the detector with sub-pixel shifts. In this way, the counting information of the pixel area is virtually divided into smaller areas, thus increasing the resolution of the images. As well as for the determination of the detector characteristics, I used MC simulations in order to investigate the feasibility of the application of the SSR to our preclinical SPECT system. Several phantoms and radioactive sources were simulated, in order to assess the performance of the proposed SPECT scanner. Finally, I modelled a four-headed preclinical SPECT scanner capable of the proper movements in order to obtain the SSR acquisition sequences. Each detector is based on a pixelated scintillator coupled to a low-energy tungsten collimator with parallel square holes. I used a three-dimensional volumetric mouse phantom, named DigiMouse, in order to obtain realistic images. This phantom was generated by using co-registered CT and cryosection images of a male laboratory mouse. The mouse images obtained by using the DigiMouse voxel phantom demonstrate the good capability of the system as a suitable tool for brain imaging (Chapter 6). Through this study, I finalized all revisions on the scanner necessary to meet the requirements of the SSR technique. Finally, I evaluated the performance of the system by applying the SSR on small animal imaging measurements achieving images that allowed discriminating small anatomical details of the experimental subjects with a resolution of about 1 mm (Chapter 7). In the last part of my PhD project, I validated the new SPECT system by using this technology to better characterize a recently developed rat model of ASD (Chapter 8). In particular, I studied the functionality of dopaminergic neurotransmission by performing imaging experiments in FMR1 knock-out (KO) rats, that have been validated as genetic animal model of ASD. Wild-type animals were used as controls. In conclusion, the activity performed during my three-year PhD project increased my knowledge about innovative molecular imaging systems. The experience gained can be spent in both preclinical and clinical settings for the design of hybrid systems that allow studying the brain at different levels for either morphological or functional investigations. (Chapter 9). The functional study of the brain using innovative tools might have important spin-offs on basic biomedical research, allowing to obtain essential information on its functioning and last but not least significant industrial implications. Besides this work, the potential that could be derived from this research project are manifold, such as a deeper understanding of pathogenetic mechanisms of brain disorders, their early diagnosis, and the development of potential new treatments. In this scenario, studying the brain at different levels of analysis represents a challenge for future research paving the way to molecular diagnostics and personalized medicine.