Hindered diffusion of MRI contrast agents in rat brain extracellular micro-environment assessed by acquisition of dynamic T 1 and T 2 maps B. Marty a , B. Djemaï a , C. Robic b , M. Port b , P. Robert b , J. Valette a , F. Boumezbeur a , D. Le Bihan a , F. Lethimonnier a and S. Mériaux a * The knowledge of brain tissues characteristics (such as extracellular space and tortuosity) represents valuable information for the design of optimal MR probes for specific biomarkers targeting. This work proposes a methodology based on dynamic acquisition of relaxation time maps to quantify in vivo MRI contrast agent concentration after intra- cerebral injection in rat brain. It was applied to estimate the hindered diffusion in brain tissues of five contrast agents with different hydrodynamic diameters (Dotarem W  1 nm, P846  4 nm, P792  7 nm, P904  22 nm and Gd-based emulsion  170 nm). In vivo apparent diffusion coefficients were compared with those estimated in an obstacle-free medium to determine brain extracellular space and tortuosity. At a 2 h imaging timescale, all contrast agents except the Gd-based emulsion exhibited significant diffusion through brain tissues, with characteristic times compatible with MR molecular imaging (<70 min to diffuse between two capillaries). In conclusion, our experiments indicate that MRI contrast agents with sizes up to 22nm can be used to perform molecular imaging on intra-cerebral biomarkers. Our quantification methodology allows a precise estimation of apparent diffusion coefficients, which is helpful to calibrate optimal timing between contrast agent injection and MRI observation for molecular imaging studies. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: brain tissue tortuosity; dynamic T 1 and T 2 mapping; extracellular diffusion; in vivo concentration quantification; MRI contrast agents 1. INTRODUCTION Since the early days of MRI, paramagnetic (e.g. lanthanides atoms) (1,2) and superparamagnetic (e.g. iron oxide particles) (3,4) contrast agents have been introduced to enhance the contrast of specific structures. More recently, the increase in signal-to-noise ratio provided by innovative MRI instrumentation has opened the way to the development of new contrast agents dedicated to molecular imaging applications (5,6), for which MRI sensitivity is one of the main challenges to be overcome. Considering the low concentration of biomarkers and the specific affinity of the functionalized probe with its target, one has to reach at least the nanomolar scale. To achieve this nanomolar detection threshold, several strategies seeking to significantly enhance contrast agent relaxivities (r 1 and r 2 ) have been proposed, often leading to high molecular weight particles and consequently high hydrodynamic diameters (d H ): 20–30 nm for dendrimers (7); 20–100 nm for ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (8); and up to 200 nm for liposomes or emulsions incorporating more than 10000 Gadolinium (Gd) atoms (9). These new high-sensitivity probes are commonly used to target endovascular biomarkers [such as a n b 3 integrins in tumor vessels, for example (6)], but their relatively high hydrodynamic size makes it difficult to use them for intra-cerebral biomarkers targeting. First, they are not able to naturally cross the blood–brain barrier (BBB) and reach cerebral tissues, and then, even if the BBB is passed through, their diffusion to targets can be considerably hindered because of their high molecular weight. Thus, design of such contrast agents must take into account the main properties of the media they will diffuse into. For brain disease studies, one valuable information is the apparent diffusion coefficient (ADC) of the contrast agent in the extracellular space (ECS), which differs from the free diffusion coefficient (D free ) owing to hindrance by cell membranes that impose tortuous paths to particle motion. From the measurements of ADC and D free , the ECS tortuosity can be computed to characterize this hindrance to contrast agent diffusion induced by cellular obstructions and then calibrate optimal injection doses and observation delay. Furthermore Thorne et al. have already shown that tortuosity values may be modified in some pathological brain tissues (for example after ischemia) (10). Several methods were proposed to measure molecule diffusion through cerebral tissues. The first method implies the use of specific radiotracers perfused for several hours in the ECS of anes- thetized animals (11–14). The perfused region of the brain is then * Correspondence to: S. Mériaux, NeuroSpin, I 2 BM, Commissariat à l’Énergie Atomique, Gif-sur-Yvette, France. E-mail: sebastien.meriaux@cea.fr a B. Marty, B. Djemaï, J. Valette, F. Boumezbeur, D. Le Bihan, F. Lethimonnier, S. Mériaux NeuroSpin, I 2 BM, Commissariat à l’Énergie Atomique, Gif-sur-Yvette, France b C. Robic, M. Port, P. Robert Research Division, Guerbet, Roissy-Charles de Gaulle, France Full Paper Received: 6 January 2012, Revised: 12 June 2012, Accepted: 4 July 2012, Published online in Wiley Online Library: 2012 (wileyonlinelibrary.com) DOI: 10.1002/cmmi.1489 Contrast Media Mol. Imaging 2013, 8 12–19 Copyright © 2012 John Wiley & Sons, Ltd. 12