Characterization of a Calcium-responsive MR-Agent in Cellular Model Systems

Introduction Calcium is an essential metal ion for life. In the brain, essential intracellular signaling processes are known to depend on Ca2+ influx from the extracellular space. Any substantial fluctuation in extracellular Ca2+ concentrations is likely to have important functional effects. Hence, the ability to non-invasively observe these changes in Ca2+ concentrations using MRI would be of paramount importance for biological research. Our recently developed smart contrast agent (SCA) showed remarkable longitudinal relaxivity changes upon interaction with Ca2+ in buffer and biologically relevant solutions, such as a model of an extracellular matrix[1]. Presently, our goal is to study the potential MR-response of our SCA in cellular model systems, as well as to characterize its physiological effects on cells and intracellular Ca2+. Methods We have designed a model system that mimics living tissues in regards to density and partially occupied (extracellular) volume by using monodisperse polystyrene microspheres. Furthermore, growing fibroblast cells (3T3) as 3D culture embedded in an extracellular matrix gel (Matrigel) were used. Microspheres were mixed or cells were perfused with a cell culture medium containing SCA. Next, we manipulated the extracellular Ca2+ concentration and determined T1 with inversion recovery experiments in an NMR spectrometer at physiological temperature. Furthermore, we monitored changes in intracellular Ca2+ and ATP-induced Ca2+ transients upon the addition of SCA to primary glial cells (conventional 2D culture of astrocytes) and checked for concentration dependence in experimental perfusion chambers. For these experiments, we used laser scanning confocal microscopy imaging using Ca2+ sensitive fluorescent probes. Results and conclusions Inversion recovery experiments revealed a change in T1 of a few milliseconds upon the alteration of Ca2+ concentrations that were comparable to changes induced during neural activity. Physiological experiments using confocal fluorescence microscopy showed that SCA reduces intracellular Ca2+ concentration upon its addition and influences ATP-induced Ca2+ transients in astrocytes. However, the response to ATP recovers after waiting for 10 minutes. Moreover, SCA is not toxic at the applied concentrations (up to 1.8 mM) and its presence does not interfere significantly with intracellular Ca2+ signaling. Overall, the 3D cellular model demonstrates that the T1-response of our SCA to extracellular Ca2+ fluctuations would be sufficient to allow the development of an entirely new in vivo fMRI method that is not based on the BOLD signal