RESUMEN
Transmembrane water permeability changes occur after initialization of necrosis and are a mechanism for early detection of cell death. Filter-exchange spectroscopy (FEXSY) is sensitive to transmembrane water permeability and enables its quantification by magnetic resonance via the apparent exchange rate (AXR). In this study, we investigate AXR changes during necrotic cell death. FEXSY measurements of yeast cells in different necrotic stages were performed and compared with established fluorescence cell death markers and pulsed gradient spin echo measurements. Furthermore, the influence of T2 relaxation on AXR was examined in a two-compartment system. The AXR of yeast cells increased slightly after incubation with 20% isopropanol, whereas it peaked sharply after incubation with 25% isopropanol. At this point, almost all the yeast cells were vital but showed compromised membranes. After incubation with 30% isopropanol, AXR measurements showed high variability, at a point corresponding to a majority of the yeast cells being in late-stage necrosis with disrupted cell membranes. Simulations revealed that, for FEXSY measurements in a two-compartment system, a long filter echo time (TEf), compared with the T2 of the slow-diffusing compartment, filters out a fraction of the slow-diffusing compartment signal and leads to overestimation of apparent diffusion coefficient (ADC) and underestimation of AXR. Our results demonstrate that AXR is sensitive to gradual permeabilization of the cell membrane of living cells in different permeabilization stages without exogenous contrast agents. AXR measurements were sensitive to permeability changes induced by relatively low concentrations of isopropanol, at levels for which no measurable effect was detectable by ADC measurements. TEf may act as a signal filter that affects the estimated AXR value of a system consisting of a variety of local diffusivities and a range of T2 that includes T2 values shorter or comparable with the TEf.
RESUMEN
OBJECTIVES: Development of a protocol for validation and quality assurance of filter-exchange imaging (FEXI) pulse sequences with well-defined and reproducible phantoms. MATERIALS AND METHODS: A FEXI pulse sequence was implemented on a 7 T preclinical MRI scanner. Six experiments in three different test categories were established for sequence validation, demonstration of the reproducibility of phantoms and the measurement of induced changes in the apparent exchange rate (AXR). First, an ice-water phantom was used to investigate the consistency of apparent diffusion coefficient (ADC) measurements with different diffusion filters. Second, yeast cell phantoms were utilized to validate the determination of the AXR in terms of repeatability (same phantom and session), reproducibility (separate but comparable phantoms in different sessions) and directionality of diffusion encodings. Third, the yeast cell phantoms were, furthermore, used to assess potential AXR bias because of altered cell density and temperature. In addition, a treatment experiment with aquaporin inhibitors was performed to evaluate the influence of these compounds on the cell membrane permeability in yeast cells. RESULTS: FEXI-based ADC measurements of an ice-water phantom were performed for three different filter strengths, showed good agreement with the literature value of 1.099 × 10-3 mm2/s and had a maximum coefficient of variation (CV) of 0.55% within the individual filter strengths. AXR estimation in a single yeast cell phantom and imaging session with five repetitions resulted in an overall mean value of (1.49 ± 0.05) s-1 and a CV of 3.4% between the chosen regions of interest. For three separately prepared phantoms, AXR measurements resulted in a mean value of (1.50 ± 0.04) s-1 and a CV of 2.7% across the three phantoms, demonstrating high reproducibility. Across three orthogonal diffusion directions, a mean value of (1.57 ± 0.03) s-1 with a CV of 1.9% was detected, consistent with isotropy of AXR in yeast cells. Temperature and AXR were linearly correlated (R2 = 0.99) and an activation energy EA of 37.7 kJ/mol was determined by Arrhenius plot. Furthermore, a negative correlation was found between cell density (as determined by the reference ADC/fe) and AXR (R2 = 0.95). The treatment experiment resulted in significantly decreased AXR values at different temperatures in the treated sample compared to the untreated control indicating an inhibiting effect. CONCLUSIONS: Using ice-water and yeast cell-based phantoms, a protocol for the validation of FEXI pulse sequences was established for the assessment of stability, repeatability, reproducibility and directionality. In addition, a strong dependence of AXR on cell density and temperature was shown. As AXR is an emerging novel imaging biomarker, the suggested protocol will be useful for quality assurance of AXR measurements within a study and potentially across multiple sites.