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Abstract

Introduction

Real-time monitoring of biological processes under physiological and pathological conditions is still great challenge for magnetic resonance imaging (MRI), despite its extensive clinical applications. Calcium is involved in an immense number of signaling events in the brain, hence it is an ideal target for functional MRI purposes. For instance, its extracellular concentration substantially varies during the ischemic stroke [1, 2]. Thus, possibility to track Ca2+ noninvasively would deepen the understanding of numerous physiological processes and allow direct monitoring of neuronal activity.

Methods

To monitor Ca2+ changes in vivo, an ischemic stroke model (transient remote MCAo-middle cerebral artery occlusion) was used. Gd2L1 (responsive probe) and Gd2L2 (control probe) were continuously infused intracranially in Wistar rats using osmotic pump, and the MCAo was induced remotely. Functional MRI measurements were divided in three segments: pre-ischemia, ischemia, and reperfusion and consisted of T1w image acquisition every 2 min. Existence of ischemia was confirmed with standard diffusion weighted imaging. Control MRI experiments included intracranial injection of Gd2L1 and Gd2L2, without causing MCAo. Data analysis was based on K-means clustering applied to the raw signals, while the data detrending was done using 4th order spline fitted to the pre-ischemia and reperfusion segments.

Results/Discussion

T1w images and corresponding cluster maps show that cluster 1 clearly corresponds to the center of injection (Figure 1). Furthermore, T1w MRI signal of Gd2L1 varied noticeably in response to the MCAo, declining through the ischemia segment. Albeit, MRI signal enhanced by Gd2L2 is merely a consequence of the infusion of the contrast agent, and did not show any alterations due to MCAo induction or tissue reperfusion. This observation can be explained by the decrease of [Ca2+] during the ischemia and accordingly longitudinal relaxivity (r1) reduction of Gd2L1. Upon reperfusion and restoration of [Ca2+], r1 recovers and so do the initial MRI signal slope. Detrended signals, freed from the transient behavior, show even greater differentiation between MCAo experiments with Gd2L1 on one hand and the ones with Gd2L2 and controls on the other hand (Figure 2), confirming that changes in MRI signal occur only during MCAo induction with Gd2L1.

Conclusions

Here we report the successful use of calcium-responsive MRI probes in vivo for early detection and monitoring of the ischemic stroke. The potential of this molecular fMRI technique is tremendous. It could allow the visualization and mapping of neural signaling using Ca2+ as its direct indicator, supplementing the use of conventional fMRI based on BOLD signal and enabling assessment of neuronal activity in direct fashion.