Hydrophysiology NMR reveals mechanisms of steady-state water exchange in neural tissue

Water molecules exchange incessantly across cell membranes and between intracellular compartments, but the dominant steady-state transport pathways---and whether they are active or passive---remain unclear. Low-field, high-gradient diffusion exchange spectroscopy (DEXSY) nuclear magnetic resonance (NMR) measurements on viable ex vivo neonatal mouse spinal cords show that water exchange is primarily passive. The apparent exchange rate constant (AXR) depends on osmotic conditions because it reflects multiple exchange pathways, each weighted by the exchanging compartments volume fractions. A faster transmembrane path which becomes more visible with increasing extracellular fraction has a high activation energy but is ion-independent, suggesting passive transport through the phospholipid bilayer and not transport through co-transporter or channel proteins. A slower pathway which dominates when the extracelluar space is shrunken has a low activation energy, consistent with geometric exchange between intracellular environments. Moreover, we show how DEXSY can be used to non-invasively measure tonicity in tissue, and inform us about the status of the tissue millieu. These findings may also inform future translation to clinical MRI. SIGNIFICANCE STATEMENTDespite waters abundance and essential role in life, few techniques allow direct interrogation of steady-state water transport and homeostasis at the cellular level in tissue. We demonstrate that diffusion exchange spectroscopy (DEXSY) NMR fills this gap by addressing two fundamental questions: How does water exchange between tissue microenvironments under steady-state conditions, and do these processes involve active water cycling? A key result is a method to non-invasively estimate tonicity in tissue.