Biology subjects

Ravin, R.

Publications and source records attributed to Ravin, R..

7 recordsLinked to original sources

bioRxiv2025

Validation of diffusion and exchange imaging biomarkers via simultaneous real-time NMR and optical microscopy

Diffusion MRI can reflect features of tissue microstructure and homeostasis, but direct validation in living neural tissue remains challenging. Here we combine optical microscopy and NMR for real-time recording on viable ex vivo neural tissue during perturbations. Simultaneous high-temporal-resolution NMR and optical microscopy are used to monitor apparent diffusion coefficient (ADC), apparent exchange rate (AXR), intrinsic optical signal (IOS), and intracellular calcium in ex vivo neonatal mouse spinal cord during osmotic and ionic perturbations. We find that ADC correlates strongly with IOS, while AXR decreases with metrics related to depolarization. ADC and AXR are sensitive to distinct features of cellular swelling, supporting their complementary roles in probing tissue viability and function.

cell biology↗

bioRxiv2025

Passive water exchange between multiple sites can explain why apparent exchange rate constants depend on ionic and osmotic conditions in gray matter

Porous materials, such as biological tissue, often have heterogeneous microstructures where imbibed fluid experiences distinct environments on short timescales, but can exchange among different environments over long timescales. Nuclear magnetic resonance (NMR) methods such as diffusion exchange spectroscopy (DEXSY) can measure this exchange in water under steady-state and equilibrium conditions; however, modeling becomes more complex when more than two exchanging environments are involved. This complexity is particularly relevant in the central nervous system (CNS), where water diffusion and exchange at the cellular level play critical roles in homeostasis. While DEXSY can measure these processes, they may not be adequately modeled as two-site exchange between intracellular and extracellular spaces (ICS and ECS). Here we study the behavior of apparent exchange rate constants (AXR) estimated from DEXSY data numerically simulated using a three-site exchange model (3XM). The 3XM is based on gray matter microstructural characteristics, incorporating both transmembrane exchange between ECS and ICS and geometric exchange between environments within ICS where water mobility differs due to the complex architecture of neurons, glial cells, and the ECS. Inspired by the Na+/K+-ATPase pump-leak model of cell volume maintenance, the 3XM accounts for effects of osmolytes, ions, and voltage on ECS and ICS volume fraction. The model predicts a significant reduction in AXR and a smaller decrease in apparent diffusion coefficients (ADC) following the level of membrane depolarization expected from Na+/K+-ATPase inhibition. These changes were reversed by the addition of membrane-impermeable ECS osmolytes, independent of voltage, in agreement with previous experiments. While the exchange rate constants for each pathway simply follow first-order kinetics, the AXRs sensitivity to these pathways depends on the ECS volume fraction. When ECS is present, transmembrane exchange dominates, but when cells swell following pump inhibition, geometric exchange becomes the dominant pathway.

cell biology↗

bioRxiv2024

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 that becomes more visible with increasing extracellular space (ECS) fraction has a high activation energy but is ion-independent, suggesting passive transport through the phospholipid bilayer but not active or passive transport through co-transporter or channel proteins. A slower pathway which dominates when the extracelluar space shrinks 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 milieu. These findings may inform future translation to clinical MRI. SignificanceWe use advanced nuclear magnetic resonance methods to address two unanswered questions in cellular biology: How does water exchange between tissue microenvironments under steady-state conditions, and do these processes involve active water cycling?

biophysics↗

bioRxiv2024

The Diffusion Exchange Ratio (DEXR): A minimal sampling of diffusion exchange spectroscopy to probe exchange, restriction, and time-dependence

Water exchange is increasingly recognized as an important biological process that can affect the study of biological tissue using diffusion MR. Methods to measure exchange, however, remain immature as opposed to those used to characterize restriction, with no consensus on the optimal pulse sequence(s) or signal model(s). In general, the trend has been towards data-intensive fitting of highly parameterized models. We take the opposite approach and show that a judicious sub-sample of diffusion exchange spectroscopy (DEXSY) data can be used to robustly quantify exchange, as well as restriction, in a data-efficient manner. This sampling produces a ratio of two points per mixing time: (i) one point with equal diffusion weighting in both encoding periods, which gives maximal exchange contrast, and (ii) one point with the same total diffusion weighting in just the first encoding period, for normalization. We call this quotient the Diffusion EXchange Ratio (DEXR). Furthermore, we show that it can be used to probe time-dependent diffusion by estimating the velocity autocorrelation function (VACF) over intermediate to long times ([~] 2 - 500 ms). We provide a comprehensive theoretical framework for the design of DEXR experiments in the case of static or constant gradients. Data from Monte Carlo simulations and experiments acquired in fixed and viable ex vivo neonatal mouse spinal cord using a permanent magnet system are presented to test and validate this approach. In viable spinal cord, we report the following apparent parameters from just 6 data points:{tau} k = 17 {+/-} 4 ms, fNG = 0.71 {+/-} 0.01, Reff = 1.10 {+/-} 0.01 m, and keff = 0.21 {+/-} 0.06 m/ms, which correspond to the exchange time, restricted or non-Gaussian signal fraction, an effective spherical radius, and permeability, respectively. For the VACF, we report a long-time, power-law scaling with {approx} t- 2.4, which is approximately consistent with disordered domains in 3-D. Overall, the DEXR method is shown to be highly efficient, capable of providing valuable quantitative diffusion metrics using minimal MR data.

biophysics↗

bioRxiv2023

"Tumor Treating Fields" delivered via electromagnetic induction have varied effects across glioma cell lines and electric field amplitudes

Previous studies reported that alternating electric fields (EFs) in the intermediate frequency (100 - 300 kHz) and low intensity (1 - 3 V/cm) regime -- termed "Tumor Treating Fields" (TTFields) -- have a specific, anti-proliferative effect on glioblastoma multiforme (GBM) cells. However, the mechanism(s) of action remain(s) incompletely understood, hindering the clinical adoption of treatments based on TTFields. To advance the study of such treatment in vitro, we developed an inductive device to deliver EFs to cell cultures which improves thermal and osmolar regulation compared to prior devices. Using this inductive device, we applied continuous, 200 kHz electromagnetic fields (EMFs) with a radial EF amplitude profile spanning 0 - 6.5 V/cm to cultures of primary rat astrocytes and several human GBM cell lines -- U87, U118, GSC827, and GSC923 -- for a duration of 72 hours. Cell density was assessed via segmented pixel densities from GFP expression (U87, U118) or from staining (astrocytes, GSC827, GSC923). Further RNA-Seq analyses were performed on GSC827 and GSC923 cells. Treated cultures of all cell lines exhibited little to no change in proliferation at lower EF amplitudes (0 - 3 V/cm). At higher amplitudes (> 4 V/cm), different effects were observed. Apparent cell densities increased (U87), decreased (GSC827, GSC923), or showed little change (U118, astrocytes). RNA-Seq analyses on treated and untreated GSC827 and GSC923 cells revealed differentially expressed gene sets of interest, such as those related to cell cycle control. Up- and down-regulation, however, was not consistent across cell lines nor EF amplitudes. Our results indicate no consistent, anti-proliferative effect of 200 kHz EMFs across GBM cell lines and thus contradict previous in vitro findings. Rather, effects varied across different cell lines and EF amplitude regimes, highlighting the need to assess the effect(s) of TTFields and similar treatments on a per cell line basis.

cancer biology↗

bioRxiv2022

Water exchange rates measure active transport and homeostasis in neural tissue

For its size, the brain is the most metabolically active organ in the body. Most of its energy demand is used to maintain stable homeostatic physiological conditions. Altered homeostasis and active states are hallmarks of many diseases and disorders. Yet there is currently no reliable method to assess homeostasis and absolute basal activity or activity-dependent changes non-invasively. We propose a novel, high temporal resolution low-field, high-gradient diffusion exchange NMR method capable of directly measuring cellular metabolic activity via the rate constant for water exchange across cell membranes. Using viable ex vivo neonatal mouse spinal cords, we measure a component of the water exchange rate which is active, i.e., coupled to metabolic activity. We show that this water exchange rate is sensitive primarily to tissue homeostasis and viability and provides distinct functional information in contrast to the Apparent Diffusion Coefficient (ADC), which is sensitive primarily to tissue microstructure but not activity. SIGNIFICANCE STATEMENTDespite what physiology text-books may report, water transport across membranes is not only a passive process. However, current understanding is limited because standard techniques can only measure net flux (the difference between water moving in and water moving out). Even so, water is constantly exchanging between the inside and outside of cells and organelles without net flux during homeostasis. We developed a Magnetic Resonance method able to "see" water molecules exchanging on shorter timescales than could be observed before. In neural tissue we find most water exchange is active, that is, linked to ATP-driven processes. This method may one day be translated to clinical MRI applications for measuring cellular function and activity in the human brain and body.

neuroscience↗

bioRxiv2019

Magnetic Resonance measurements of sub-cellular membrane structures in live and fixed neural tissue

We develop magnetic resonance (MR) methods for measuring real-time changes of tissue microstructure and membrane permeability of live and fixed neural tissue. Diffusion and exchange MR measurements are performed using the large static gradient produced by a single-sided permanent magnet. Using tissue delipidation methods, we show that water diffusion is restricted solely by lipid membranes. Most of the diffusion signal can be assigned to water in tissue which is far from membranes. The remaining 25% can be assigned to water restricted on length scales of roughly a micron or less, near or within membrane structures at the cellular, organelle, and vesicle levels. Diffusion exchange spectroscopy measures water exchanging between membrane structures and free environments at 100 s-1.

biophysics↗