Investigation of a Calcium-Responsive Contrast Agent in Cellular Model Systems: Feasibility for Use as a Smart Molecular Probe in Functional MRI

Abstract

Responsive or smart contrast agents (SCAs) represent a promising direction for development of novel functional MRI (fMRI) methods for the eventual noninvasive assessment of brain function. In particular, SCAs that respond to Ca 2+ may allow tracking neuronal activity independent of brain vasculature, thus avoiding the characteristic limitations of current fMRI techniques. Here we report an in vitro proof-ofprinciple study with a Ca 2+ -sensitive, Gd 3+ -based SCA in an attempt to validate its potential use as a functional in vivo marker. First, we quantified its relaxometric response in a complex 3D cell culture model. Subsequently, we examined potential changes in the functionality of primary glial cells following administration of this SCA. Monitoring intracellular Ca 2+ showed that, despite a reduction in the Ca 2+ level, transport of Ca 2+ through the plasma membrane remained unaffected, while stimulation with ATP induced Ca 2+ -transients suggested normal cellular signaling in the presence of low millimolar SCA concentrations. SCAs merely lowered the intracellular Ca 2+ level. Finally, we estimated the longitudinal relaxation times (T 1 ) for an idealized in vivo fMRI experiment with SCA, for extracellular Ca 2+ concentration level changes expected during intense neuronal activity which takes place upon repetitive stimulation. The values we obtained indicate changes in T 1 of around 1−6%, sufficient to be robustly detectable using modern MRI methods in high field scanners. Our results encourage further attempts to develop even more potent SCAs and appropriate fMRI protocols. This would result in novel methods that allow monitoring of essential physiological processes at the cellular and molecular level.

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“…1 MR probes capable of detecting specific biological events, including changes in pH, 2,3 ion concentration, 4–6 and the activity of enzymes 7 have been developed to expand MRI’s capabilities. Responsive MR agents are often based on modulating magnetic effects on the 1 H nuclei of water.…”

Dendrimeric calcium-responsive MRI contrast agents with slow in vivo diffusion

“…1). 16,17 Moreover, studies in a complex cell culture model suggest that the T 1 change in vivo may exceed several percent, more than the current fMRI methodology allows. an increase in the molecular weight), although the responsive part of the SCA must be retained, as well as its Ca-sensitivity and MRI activity.…”

“…16 We therefore selected smaller ROIs closer to the injection site where higher SCA concentrations would be expected shortly after the intracerebral injection (40-90 min). The maximal SI changes in dynamic, T 1 -weighted fMRI experiments based on this concept are expected in regions with higher or similar SCA concentration relative to the local extracellular Ca-concentration ([SCA] Z [Ca 2+ ] e ).…”

MRI‐based Sensors for In Vivo Imaging of Metal Ions in Biology

“…Although the above mentioned strategies, and, recently, others, have shown the potential in designing Ca 2+ ‐responsive MRI T 1 ‐contrast agents, only a few examples have demonstrated the performance of such platforms in complex biological systems either in vitro or in vivo . A calcium sensor based on an EGTA‐binder linked to Gd 3+ ‐DO3A complexes showed the ability to monitor changes in extracellular Ca 2+ levels in the vicinity of live cells . In this work, a cell culture model was used to demonstrate the feasibility of the proposed EGTA‐Gd‐DO3A complex to monitor (54 % increase in r 1 , values changed from 2.45 mM −1 s −1 to 3.79 mM −1 s −1 at 7.0T MRI) the expected changes in extracellular Ca 2+ levels upon neuronal activation while mimicking real physiological conditions.…”