Measurements and simulations of transmembrane water exchange by diffusion NMR methods: From yeast cells to optic nerve ex vivo

Non-invasive measurements of exchange is paramount in different fields, ranging from material to biological sciences, and may even blur micro-structural or other characteristics of multi compartmental systems studied by MR methods. Despite the growing interest in diffusion-exchange studies of complex systems—where at least one exchanging component exhibits non-Gaussian diffusion—comparative studies remain scarce. Most existing investigations have applied different diffusion MR methods to different biological samples under varying experimental conditions, making direct comparisons difficult. Moreover, the lack of a gold standard for exchange rate measurements further complicates efforts to validate and interpret results. To address these challenges, we employed two diffusion NMR-based methods—the constant-gradient pulsed field gradient (CG-PFG) and the recently introduced filter-exchange NMR spectroscopy (FEXSY)—to investigate apparent water exchange in yeast cells and optic nerves, both before and after fixation. We first evaluated the effect of the q-values on the extracted indices and then evaluated the stability and reproducibility of the measurements. The CG-PFG and FEXSY experiments were collected on the same sample to allow for comparison of the results. The intracellular mean residence times (MRTs) ( τ _i ) extracted from the log-linear fit of the CG-PFG NMR experiments were found to be 554± 6ms and 337 ± 10ms for yeast cells before and after fixation, respectively. The respective τ _i values extracted from the FEXSY experiments before and after fixation were found to be 368± 14ms and 146± 24ms, respectively. Despite the difference in absolute values of the MRTs, the same qualitative behavior is observed in the two methodologies, and both could be analyzed using the bi-compartmental Kärger model. The same methodologies were then used to study exchange in the more complex porcine optic nerves. There, the bicompartmental Kärger model analysis is shown to be inadequate. Extensive Monte Carlo simulations are used to narrow down on the most possible explanation, suggesting that optic nerves are multi-compartmental systems where not all spins are free to undergo exchange. Supporting theoretical calculation point to the existence of at least one additional non-exchanging restricted compartment. Thus, a tri-compartmental model is derived and used to analyze the data. The new model fits the data significantly better and results in dramatically different exchange rates when used on white matter (WM) data: CG-PFG experiments were found to be 730 ± 40ms and 803 ± 16ms for optic nerve before and after fixation, respectively. The respective τ _i values extracted from the FEXSY experiments before and after fixation were found to be 530 ± 125ms and 387 ± 104ms, respectively. These values are considerably smaller than values that were previously reported. Finally, we use simulations to show that the quantitative discrepancy between the CG-PFG and FEXSY can be attributed, at least partially, to the difference in T ₂ values between the intra- and extracellular compartments. We thus encourage the pairing of exchange and spin-spin relaxation measurements in future works. We end with a discussion on the current state of the diffusion-exchange, and in an attempt to put a spotlight on essential corner stones that are still missing despite the great advance of recent years — experimental standardization, method comparison and adequate modeling.