Awake Rat Brain Functional Magnetic Resonance Imaging Using Standard Radio Frequency Coils and a 3D Printed Restraint Kit

Functional magnetic resonance imaging (fMRI) is a powerful noninvasive tool for studying spontaneous resting state functional connectivity (RSFC) in laboratory animals. Brain function can be significantly affected by generally used anesthetics, however, rendering the need for awake imaging. Only a few different awake animal habituation protocols have been presented, and there is a critical need for practical and improved low-stress techniques. Here we demonstrate a novel restraint approach for awake rat RSFC studies. Our custom-made 3D printed restraint kit is compatible with a standard Bruker Biospin MRI rat bed, rat brain receiver coil, and volume transmitter coil. We also implemented a progressive habituation protocol aiming to minimize the stress experienced by the rats, and compared RSFC between awake, lightly sedated, and isoflurane-anesthetized rats. Our results demonstrated that the 3D printed restraint kit was suitable for RSFC studies of awake rats. During the short 4-day habituation period, the plasma corticosterone concentration, movement, and heart rate, which were measured as stress indicators, decreased significantly, indicating adaptation to the restraint protocol. Additionally, 10 days after the awake MRI session, rats exhibited no signs of depression or anxiety based on open-field and sucrose preference behavioral tests. The RSFC data revealed significant changes in the thalamo-cortical and cortico-cortical networks between the awake, lightly sedated, and anesthetized groups, emphasizing the need for awake imaging. The present work demonstrates the feasibility of our custom-made 3D printed restraint kit. Using this kit, we found that isoflurane markedly affected brain connectivity compared with that in awake rats, and that the effect was less pronounced, but still significant, when light isoflurane sedation was used instead.