Modulation of the intracellular endogenous chemical exchange saturation transfer (CEST) effect through extracellular gadolinium contrast agents (GdCA) has recently been proposed as a method to measure water exchange rates across cell membranes¹. In this study, we applied this novel method to investigate water cycling in vivo for the first time using the chick embryo in ovo as a model organism². The blood-brain barrier in the chick embryo is still immature, allowing GdCAs to diffuse from the bloodstream into the interstitial space of the brain parenchyma³. The presence of GdCAs in the extracellular space attenuates the intracellular CEST effect in a manner dependent on the transmembrane water exchange rate, with faster exchange leading to greater attenuation.

Experiments were conducted using a Bruker Pharmascan 7 Tesla MRI scanner.  Following the administration of 100μl of Gd-HPDO3A (0.5M) into the chorioallantonic membrane (CAM), we measured concentrations of GDCA exceeding >1 mM in brain parenchyma. To elucidate the mechanisms of water transport, transmembrane water exchange rates were measured under different physiological conditions: in the awake animal, under general anaesthesia, and post-mortem.

Our results show that transcytolemmal water exchange is faster than previously reported from ex vivo measurements in tissue preparations and is driven by activity-dependent cellular transport⁴. Furthermore, we found that general anaesthesia reduces exchange rates by more than 10-fold in both neurons and astrocytes, suggesting a significant impact on water homeostasis.

This method provides a powerful approach to investigate brain development and the influence of genetic or environmental factors during prenatal stages.

Figure 1. (a) Morphological images (sagittal view) of a chicken embryo inside the egg, obtained with a T₂w sequence acquired using 7 Tesla MRI on embryonic day 18 (E18). The red ROI indicates the area from which data for the z-spectrum were obtained, the telencephalon. The ROIs designed for signal analysis were delineated conservatively to ensure the observation of contributions solely from the telencephalon and (b) T2-weighted morphological images (coronal view) of a chicken embryo inside the egg acquired at 7 Tesla MRI on E18. The chorioallantoic membrane (CAM) is visible in the bottom right portion, corresponding to the air sac.

Figure 2. (a) Schematic representation of the three compartments (pool of exchangeable protons, intracellular water, extracellular water). Gadolinium is confined to the extracellular space, where the T₁ of water protons is shortened by interactions with the paramagnetic agent. The T₁ of the intracellular water is affected by extracellular gadolinium due to water exchange across the membrane. Hence, the intracellular water T₁ depends on the trancytolemmal water exchange rate k_H2O. The CEST response from the intracellular water and the pool of exchangeable protons is strongly dependent on water T₁ and can be exploited to indirectly measure k_H2O.(b) The CEST contrast enhancement mechanism is illustrated with a 3-site exchange between a CEST pool, intracellular water, and extracellular water with GBCA (blue and light blue line) with and without GBCA (red line). Asymmetry analysis (MTRasym) is performed by subtracting the water signal from one side of the z-spectrum from the other side to emphasize the effects of chemical exchange (dotted lines). The more rapidly water exchanges across the membranes, the stronger the effect of extracellular Gd on the intracellular CEST effect. The dependence on transmembrane water exchange rate is shown by comparing the two curves where GBCA is present in the extracellular compartment. Under otherwise identical conditions, the slower exchange is represented by the light blue line, while the faster exchange is depicted by the blue line.

References

1 Di Gregorio E., Papi C., Conti L., Di Lorenzo A., Cavallari E., Salvatore M., Cavaliere C., Ferrauto G., Aime S., “A Magnetic Resonance Imaging – Chemical Exchange Saturation Transfer (MRI-CEST) method for the detection of water cycling across cellular membranes”, Angewandte Chemie, 2023. DOI: 10.1002/anie.202313485

2 Lorenzi E., Tambalo S., Vallortigara G., Bifone A., “Manganese-enhanced magnetic resonance imaging reveals light-induced brain asymmetry in embryo”, eLife, 2023. DOI: 10.7554/eLife.86116

3 Zosen D., Hadera M. G., Lumor J. S., Andersen J. M., Paulsen R. E., “Chicken embryo as animal model to study drug distribution to the developing brain”, Journal of Pharmacological and Toxicological Methods, 112 (2021). DOI: 10.1016/j.vascn.2021.107105

4 Cavallari E., Lorenzi E., Di Gregorio E., Ferrauto G., Aime S., Vallortigara G., Bifone A., “In vivo assessment of the influence of general anaesthetics on transmembrane water cycling in the brain”, Journal of Cerebral Blood Flow & Metabolism, 2024. DOI: 10.1177/0271678X241309783

Acknowledgements

The MR imaging work was supported by FOE contribution to the EuroBioImaging MultiModal Molecular Imaging Italian Node (www.mmmi.unito.it), the PNRR PoC Nodes REDiRECt-Gd Project and the PNRR PoC Nodes ROSEWATER Project. Giorgio Vallortigara acknowledges support from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement SPANUMBRA No. 833504).