Ischemia‐induced changes of intracellular water diffusion in rat glioma cell cultures

Diffusion‐weighted MRI is commonly used in the diagnosis and evaluation of ischemic stroke because of the rapid decrease observed in the apparent diffusion coefficient (ADC) of tissue water following ischemia. Although this observation has been clinically useful for many years, the biophysical mechanisms underlying the reduction of tissue ADC are still unknown. To help elucidate these mechanisms, we have employed a novel three‐dimensional (3D) hollow‐fiber bioreactor (HFBR) perfused cell culture system that enables cells to be grown to high density and studied via MRI and MRS. By infusing contrast media into the HFBR, signals from intracellular water and extracellular water are spectroscopically resolved and can be investigated individually. Diffusion measurements carried out on C6 glioma HFBR cell cultures indicate that ischemia‐induced cellular swelling results in an increase in the ADC of intracellular water from 0.35 μm²/ms to approximately 0.5 μm²/ms (diffusion time = 25 ms). Magn Reson Med 60:258–264, 2008. © 2008 Wiley‐Liss, Inc.     

Intracellular Volume Measurement and Detection of Edema: Multinuclear NMR Studies of Intact Rat Hearts during Normothermic Ischemia

The present study describes the cell volume dynamics in intact rat hearts, during ischemia and after reperfusion. Cell volumes were measured in isolated hearts by either ¹³C or ⁵⁹Co NMR of mannitol or cobalticyanide, respectively, as extracellular markers and ¹H NMR of water as the aqueous space marker. A constant volume chamber was built inside a 15-mm NMR tube; the contents of the chamber were measured with and without a heart. The intracellular volume of isolated rat hearts was estimated to be 2.45 ± 0.13 ml/g dry weight. In the perfused heart, adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations were calculated to be 12.2 ± 0.7 and 16.1 ± 1.0 mM, respectively. Consecutive volume measurements showed cell swelling of 16% during 30 min of ischemia, which was reduced at reperfusion to 7%. After 30 min of reperfusion, ATP and PCr concentrations were 4.5 ± 0.8 and 8.1 ± 0.9 mM. It is concluded that: (1) cell swelling is an ischemic event, which is partially reversed by reperfusion; and (2) continuous measurement of cell volumes provides intracellular molar concentrations of metabolites, which are the physiologically significant parameters.     

Modulation of water diffusion during gonadotropin-induced ovulation: nmr microscopy of the ovarian follicle

The preovulatory rat follicle reaches a diameter of 1 mm with no internal blood vessels. Nutrient supply to the enclosed oocyte depends solely on passive diffusion across the follicular wall and the follicular fluid. Spin-echo and stimulatedecho NMR microscopy experiments were applied here for studying modulations in water diffusion during gonadotropin-induced maturation of perfused rat ovarian follicles (32°C). Two diffusion compartments were observed for the follicular wall. The intracellular water diffusion coefficient, measured at a short diffusion time (9 ms) was 0.28 x 10^?5 cm²/s. Diffusion at long diffusion times was restricted to 16 μm, the size of cells in the follicular wall, and did not change during maturation. In the follicular fluid a transient 26% decrease in the diffusion coefficient was observed 4–7 h after gonadotropin stimulation, a change that is bound to affect the metabolic balance of the oocyte before ovulation.     

In vitro proton NMR study of collagen in human aortic wall

The authors relate the findings in the ¹H solid state line shape (at 60 MHz) of human aortic walls (n = 12) in native state and after histologically controlled selective lysis of collagen and elastin. An analysis of the line shape shows a composite free induction decay (FID) consisting of a low amplitude (3-7%) fast decaying component (T₂ = 20 us) and a slow decaying one (T₂ > 1 ms). The fast component is identified as the protons of the collagen macromolecules. The second moment computed from the experimental fast component of the FID is in agreement with published studies examining the motional characteristics of collagen by multinuclear NMR employing spin labeling. A theoretical second moment is computed for the collagen macromolecular backbone from the atomic positions in the superhelix. Comparison with the observed experimental values allows determination of the step angle (29°) of the fast rotational motion of the collagen strands along their long axis.     

Intracellular water specific MR of microbead-adherent cells: HeLa cell intracellular water diffusion.

The (1)H MR signal arising from flowing extracellular media in a perfused, microbead-adherent cultured cell system can be suppressed with a slice-selective, spin-echo pulse sequence. The signal from intracellular water can, thus, be selectively monitored. Herein, this technique was combined with pulsed field gradients (PFGs) to quantify intracellular water diffusion in HeLa cells. The intracellular water MR diffusion-signal attenuation at various diffusion times was well described by a biophysical model that characterizes the incoherent displacement of intracellular water as a truncated Gaussian distribution of apparent diffusion coefficients (ADCs). At short diffusion times, the water "free" diffusion coefficient and the surface-to-volume ratio of HeLa cells were estimated and were, 2.0 +/- 0.3 microm(2)/ms and 0.48 +/- 0.1 microm(-1) (mean +/- SD), respectively. At long diffusion times, the cell radius of 10.1 +/- 0.4 microm was inferred and was consistent with that measured by optical microscopy. In summary: 1) intracellular water "free" diffusion in HeLa cells was rapid, two-thirds that of pure water; and 2) the cell radius inferred from modeling the incoherent displacement of intracellular water by a truncated Gaussian distribution of ADCs was confirmed by independent optical microscopy measures.     

Microscopic diffusivity compartmentation in formalin‐fixed prostate tissue

MR microimaging at 16.4 T with 40‐μm isotropic voxels was used to investigate compartmentation of water diffusion in formalin‐fixed prostate tissue. Ten tissue samples (～ 28 mm³ each) from five organs were imaged. The mean diffusivity of epithelial, stromal, and ductal/acinar compartments was estimated by two methods: ( 1 ) manual region of interest selection and ( 2 ) Gaussian fitting of voxel diffusivity histograms. For the region of interest‐method, the means of the tissue sample compartment diffusivities were significantly different (P < 0.001): 0.54 ± 0.05 μm²/ms for epithelium‐containing voxels, 0.91 ± 0.17 μm²/ms for stroma, and 2.20 ± 0.04 μm²/ms for saline‐filled ducts. The means from the histogram method were also significantly different (P < 0.001): 0.45 ± 0.08 μm²/ms for epithelium‐containing voxels, 0.83 ± 0.16 μm²/ms for stroma, 2.21 ± 0.02 μm²/ms for duct. Estimated partial volumes of epithelial, stromal, and ductal/acinar compartments in a “tissue only” subvolume of each sample were significantly different (P < 0.02) between cancer and normal tissue for all three compartments. It is concluded that the negative correlation between apparent diffusion coefficient and cancer Gleason grade observed in vivo results from an increase of partial volume of epithelial tissue and concomitant decrease of stromal tissue and ductal space. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.     

Influence of cell cycle phase on apparent diffusion coefficient in synchronized cells detected using temporal diffusion spectroscopy

The relationship between the apparent diffusion coefficient of tissue water measured by MR methods and the physiological status of cells is of particular relevance for better understanding and interpretation of diffusion‐weighted MRI. In addition, there is considerable interest in developing diffusion‐dependent imaging methods capable of providing novel information on tissue microstructure, including intracellular changes. To this end, both the conventional pulsed gradient spin–echo methods and the oscillating gradient spin–echo method, which probes diffusion over very short distance (<<cell size) and time scales, were used to measure apparent diffusion coefficient of synchronized packed HL‐60 cells at 7 T. The results show that the pulsed gradient spin–echo method with relatively long diffusion times does not detect changes in apparent diffusion coefficient when structural variations arise during cell division. On the contrary, the oscillating gradient spin–echo method can detect and quantify major changes in intracellular organization that occur during mitosis by appropriate choice of gradient frequency. Cell structural parameters, including cell size, intracellular diffusion coefficient, and surface‐to‐volume ratio were also obtained by fitting the oscillating gradient spin–echo data to simple analytical models. These oscillating gradient spin–echo features may be used in diffusion‐weighted MRI to create parametric maps that may be useful for detecting cancer or changes caused by treatment. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Quantitative nmr microscopy of multicellular tumor spheroids and confrontation cultures

In cancer research, tumor spheroids are a well established system to study tumor metabolism resembling the situation in vivo more closely cell monolayers. Spherical aggregates of malignant melanoma cells (MV3) and their invasion into rat brain aggregates have been investigated by quantitative NMR microscopy. Relaxation times (T₁, T₂) and diffusion parameter images were acquired with an in-plane resolution of 14 × 14 μm². The authors were able to demonstrate that the morphology of the spheroids can be visualized on these NMR maps. The contrast was mainly manifested in relaxation maps, where average relaxation times T₁ = 1.94 ± 0.17 s and T₂ = 42.8 ± 6.3 ms were obtained for proliferating cells, and T₁ = 2.49 ± 0.31 s and T₂ = 104.3 ± 29.4 ms for the necrobiotic center. The mean diffusion coefficients were 0.59 ± 0.12 μm²/ms and 0.85 ± 0.14 μm²/ms, respectively. The authors could follow the dynamic process of tumor cell invasion in the investigated co-culture system. Knowledge about tumor cell migration and tumor cell invasion is essential for the understanding of cancer and its therapy. Quantitative NMR microscopy can study this dynamic process noninvasively and therefore may help to assess the influence of therapy on the micromilieu of these spheroids.     

Determination of the intracellular sodium concentration in perfused mouse liver by 31P and 23Na magnetic resonance spectroscopy

A combination of ³¹P and ²³Na NMR spectroscopy has been used to quantify the concentration of intracellular sodium, [Na]_IC in the isolated and perfused mouse liver. The ³¹P resonances of dimethyl methylphosphonate and LaDOTP^5?, markers of total tissue space and extracellular space, respectively, were used to determine the intracellular liver volume. For a mean wet weight of 1.7 ± 0.3 g, the intracellular liver volume as measured by ³¹P NMR averaged 1.2 ± 0.2 ml. The amount of intracellular sodium was measured from the baseline-resolved intracellular ²³Na resonance during perfusion of the shift reagent, TmDOTP^5?. These two measurements resulted in an NMR-determined value for [Na]_IC of 29.0 ± 5.2 mM. Separate measurement of total tissue Tm and Na by atomic absorption spectroscopy on the same samples provided an AAS-determined value for [Na]_Ic of 32.1 ± 7.4 mM. These results indicate that intracellular sodium in the isolated, perfused liver is 100% visible by ²³Na NMR spectroscopy.     

Intracellular and extracellular spaces and the direct quantification of molar intracellular concentrations of phosphorus metabolites in the isolated rat heart using 31P NMR spectroscopy and phosphonate markers

To quantify metabolite and cation concentrations using NMR spectroscopy, the volumes of intracellular and extracellular spaces must be known. We describe a simple ³¹P NMR spectroscopic method that employs dimethyl methylphosphonate (DMMP) as a marker of total water space and phenylphosphoriate (PPA) as a marker of extracellular space to determine intracellular and extracellular space volumes in the isolated, perfused rat heart. In vivo and in vitro radiolabel studies were used to verify this method. The difference between the total and extracellular water spaces, determined as milliliters/heart, gave the intracellular volume and allowed direct calculation of myocardial creatine phosphate, ATP, and inorganic phosphate concentrations, which were 13.4 mM, 10.1 mM, and 3.4 mM, respectively, for the glucose-perfused rat heart. The extracellular volume decreased by 84% in hearts subjected to 28 min total, global ischemia and increased by 15% during reperfusion. The method described allows the determination of intracellular energy metabolite concentrations in perfused rat heart directly from a single, fully relaxed ³¹P NMR spectrum.     

Evidence that both fast and slow water ADC components arise from intracellular space.

Evaluation of water diffusion in the brain has revealed both fast- and slow-diffusing water populations. It has been suggested that these populations represent extra- and intracellular water, respectively. We have identified and characterized both populations in the intracellular space of the Xenopus oocyte. We have also determined their T(1) and T(2) relaxation properties. The fast and slow intracellular populations have diffusion coefficients of 1.06 +/- 0.05 microm(2)/ms and 0.16 +/- 0.02 microm(2)/ms, respectively, with the fast fraction representing 89% +/- 1% of the total water signal. These values are quite similar to those for total water in brain and are observed in the absence of signal from the perfusate (extracellular) water population. Volumetric swelling (16% +/- 4%) of the oocyte in hypoosmotic media increased the diffusion coefficients of both intracellular populations (fast = 1.27 +/- 0.03 microm(2)/ms, slow = 0.22 +/- 0.02 microm(2)/ms), but did not change their relative signal fractions. This phenomenon runs counter to the effects observed in brain injury, following which the apparent diffusion coefficient (ADC) decreases 30-50%. The results presented herein suggest that this ADC decrease in brain occurs despite cell swelling, which by itself would be expected to induce an increase in intracellular diffusion coefficients.     

Water diffusion in a rat glioma during ganciclovir-thymidine kinase gene therapy-induced programmed cell death in vivo: correlation with cell density

PURPOSE: To study the characteristics of diffusion magnetic resonance imaging (MRI) contrast in a rat brain BT4C glioma during progression of ganciclovir (GCV)-thymidine kinase gene therapy-induced programmed cell death (PCD) in vivo. MATERIALS AND METHODS: The trace of the diffusion tensor (Dav = 1/3TraceD), T2, and spin density were determined by MRI and the apparent diffusion coefficient (ADC) of water by diffusion nuclear MR (NMR) spectroscopy using largely varying b values and diffusion times (tD) at 4.7 T. Cell count and apoptotic cells were quantified by histological means. RESULTS: Decline in cell count was strongly associated with increase in both Dav and T2. Spin density ratio between tumor and contralateral parietal cortex increased with a very similar time course as Dav and T2, indicating net water gain into the eradicating tumor. Diffusion spectroscopy showed a nonmonoexponential signal decay at all tD values ranging from 14-192 msec. During PCD, the ADC of the component yielding fast diffusion coefficient (D1), as acquired with tD > or = 47 msec, increased with kinetics similar to those of Dav (tD = 4.8 msec). The fractional size of D1 increased by 10% to 15% throughout the entire tD range. Apparent water residence time of the slow diffusion component, D2, shortened from a value of 38.3 +/- 1.7 msec on day 0 to 33.4 +/- 0.5 msec by day 8. CONCLUSION: The present results show that reduced cell density and increased water content, leading to altered water microenvironment, are associated with increased water diffusion coefficient in eradicating gliomas as a result of PCD.     

Simultaneous 1H PFG-NMR and confocal microscopy of monolayer cell cultures: effects of apoptosis and necrosis on water diffusion and compartmentalization.

We induced apoptosis and necrosis in monolayer cultures of Chinese hamster ovary cells using okadaic acid and hydrogen peroxide (H2O2), respectively, and examined the effect on water diffusion and compartmentalization using pulsed-field-gradient (PFG) 1H-NMR and simultaneous confocal microscopy. In PFG experiments characterized by a fixed diffusion time (<4.7 ms) and variable b-values (0-27000 s/mm2), 1H-NMR data collected with untreated cells exhibited multiexponential behavior. Analysis with a slow-exchange model revealed two distinct cellular water compartments with different apparent diffusion coefficients (ADCs; 0.56, 0.06 x 10(-3) mm2/s) and volume fractions (0.96 and 0.04). During the first 12 hr of necrosis or apoptosis, the amount of water in the smallest compartment increased twofold before significant changes in cell density or plasma membrane integrity occurred. Over the same period, water content in the largest compartment decreased by a factor of >2 in apoptotic cells, in accordance with observed cell shrinkage, and changed little in necrotic counterparts, where only slight swelling was evident. These results indicate that PFG 1H-NMR serves as a sensitive indicator of early cell death in monolayer cultures, and can be used to distinguish apoptosis from necrosis. Measurements of restricted diffusion and water exchange are presented to elucidate the compartment origins and justify the model assumptions.     

Changes in intracellular water diffusion and energetic metabolism in response to ischemia in perfused C6 rat glioma cells

This work reports results of experiments in hollow‐fiber bioreactor C6 glioma cell cultures where the apparent diffusion coefficient (ADC) of intracellular water (iADC) was measured at diffusion times between 0.83 and 40 ms. The experiments were carried out before and after the onset of permanent ischemia. The changes in iADC following ischemia were dependent on the diffusion time employed in the experiment. An ischemia‐induced decrease in the iADC was measured at short diffusion times, while at long diffusion times the iADC increased. Decreases in the iADC measured at short diffusion times are interpreted to be a result of a decrease in the intrinsic diffusivity of intracellular water due to energy failure. Increases in iADC measured at long diffusion times, are interpreted to result from cell swelling. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

Simultaneous intracellular and extracelular pH measurement in the heart by 19F NMR of 6-fluoropyridoxol

6-Fluoropyridoxol (6-FPOL) was evaluated as a simultaneous indicator of intracellular and extracellular pH and, hence, pH gradient in perfused rat hearts. After infusion, ¹⁹F NMR spectra rapidly showed two well-resolved peaks assigned to the intracellular and extracellular compartments, and pH was calculated on the basis of chemical shift with respect to a sodium trifluoroacetate standard. To demonstrate use of this molecule, dynamic changes in myocardial pH were assessed with a time resolution of 2 min during respiratory and metabolic alkalosis or acidosis and ischemia. For a typical heart, intracellular pH (pHi) = 7.14 ± 0.01 and extracellular pH (pHe) = 7.52 ± 0.02. In response to metabolic alkalosis, pHi remained relatively constant and the pH gradient increased. In contrast, respiratory challenge caused a significant increase in pHi. Independent measurements using pH electrodes and ³¹P NMR confirmed validity of the ¹⁹F NMR results.     

Diffusion MRI and spectroscopy in Rasmussen's encephalitis

The purpose of this study was to evaluate the diffusion MRI and proton MR spectroscopy findings in a rare disorder, Rasmussen's encephalitis. Diffusion MRI studies of 3 cases of Rasmussen's encephalitis were performed using the echo-planar trace sequence (TR=5700 ms, TE=139.03 ms). The gradient-echo diffusion sequence, PSIF (TR=21.6 ms, TE=5 ms), which is a reverse FISP sequence, and proton MR spectroscopy (TR=1500 ms, TE=40 ms) were applied in two patients. The trace sequence revealed high apparent diffusion coefficient values at the diseased regions (1.21±0.13×10^–3 mm²/s), compared with normal parenchymal values in 12 control cases (0.84±0.09×10^–3 mm²/s), indicating increased motion of water molecules (disintegration of the tissue) in these regions. The PSIF sequence revealed pixel value differences between the two hemispheres. Proton MR spectroscopy revealed decreased N-acetyl aspartate peaks, compared with five control cases.     

Molecular self-diffusion of intracellular metabolites in rat brain in vivo Investigated by Localized Proton NMR Diffusion Spectroscopy

Molecular self-diffusion coefficients of water (0.75 ± 0.05), Nacetylaspartate (0.27 ± 0.04), creatines (0.27 ± 0.04), and cholines (0.28 ± 0.08) × 10^?5 cm² s^?1 were obtained from localized proton NMR spectra of rat brain in vivo using diffusion-weighted stimulated-echo (STEAM) sequences with a diffusion time of (Δ ? δ/3) = 17 ms.     

Anomalous Transverse Relaxation in 1H Spectroscopy in Human Brain at 4 Tesla

Longitudinal (T₁) and apparent transverse relaxation times (T₂) of choline-containing compounds (Cho), creatine/phospho-creatine (Cr/PCr), and N-acetyl aspartate (NAA) were measured in vivo in human brain at 4 Tesla. Measurements were performed using a water suppressed stimulated echo pulse sequence with complete outside volume presaturation to improve volume localization at short echo times. T₁-values of Cho (1.2 ± 0.1 s), Cr (1.6 ± 0.3 s), and NAA (1.6 ± 0.2 s) at 4 Tesla in occipital brain were only slightly larger than those reported in the literature at 1.5 Tesla. Thus, TR will not adversely affect the expected enhancement of signal-to-noise at 4 Tesla. Surprisingly, apparent T₂-values of Cho (142 ± 34 ms), Cr (140 ± 13 ms), and NAA (185 ± 24 ms) at 4 Tesla were significantly smaller than those at 1.5 Tesla and further decreased when increasing the mixing interval TM. Potential contributing factors, such as diffusion in local susceptibility related gradients, dipolar relaxation due to intracellular paramagnetic substances and motion effects are discussed. The results suggest that short echo time spectroscopy is advantageous to maintain signal to noise at 4 Tesla.     

Evaluation of intracellular diffusion in normal and globally-ischemic rat brain via 133Cs NMR

The question of whether the apparent diffusion coefficient (ADC) of intracellular water changes after brain injury was addressed by using ¹³³Cs as an indicator to report on the state of the intracellular environment. Cesium is an NMR-detect-able potassium analog that accumulates in the intracellular space and is detectable in rat brain after being added to the animal's diet. The ADC of cesium was measured before and after the death of the rat. The cesium ADC fell from 0.91 ± 0.05 × 10^?3 mm²/s (mean ± SEM, n = 5) in the alive rat to 0.71 ± 0.05 × 10^?3 mm²/s within 20 min (the best time resolution of the experiment) of the death of the animal and stayed at this value for at least 3 h (P < 0.001). Assuming that the ADC of cesium reflects motion in the intracellular environment, these results support the idea that there are changes associated with cell injury that would cause a reduction in the ADC of intracellular water. Hence, one factor contributing to the decrease in water ADC after brain injury is a change in the ADC of intracellular water.     

Apparent diffusion of water, ions, and small molecules in the Xenopus oocyte is consistent with Brownian displacement.

The incoherent displacement of water in living tissues is of considerable interest because of the widespread use of diffusion-weighted MRI, for which image contrast is based on the water apparent diffusion coefficient (ADC). It has been hypothesized that the decrease in water ADC associated with brain injury is primarily due to a reduction in the ADC of water in the intracellular space. Xenopus oocytes permit direct measurement of ADC values for intracellular molecules, thereby providing insight into the nature of intracellular motion. In this study, the measured ADC values of small molecules and ions are shown to be primarily size-dependent, indicating that intracellular water motion in the oocyte is mainly Brownian displacement with little or no role for cytoplasmic streaming. Further, intracellular water ADC values show no dependence on diffusion time over a broad range (3.4-100 ms), suggesting that barriers to displacement are finely spaced (< or = 2-3 microm). The water diffusion shows some small anisotropy, suggesting that the cell has structure, giving water displacement a directional preference. The calculated intracellular apparent viscosity, which reflects the combined effects of barriers to motion, intermolecular binding, and fluid phase viscosity was 2.07 +/- 0.09 cP.