Evaluation of water diffusion in the brain has revealed both fastand 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. When applied clinically, diffusion-weighted imaging is typically used to evaluate water motion over a relatively low b-value range (0 -1000 s/mm 2 ). Recently, there has been increasing interest in characterizing water motion over much larger b-value ranges (0 -10000 s/mm 2 ). Early studies over this expanded range noted that water diffusion in brain was biexponential and could be described by fast and slow diffusion coefficients. The fast population has a diffusion coefficient on the order of 1.2 m 2 /ms with a volume fraction of 70%. The slow population has a diffusion coefficient of approximately 0.2 m 2 /ms with a volume fraction of 30% (1-3). The slow diffusion coefficient offers potential as a new image contrast or clinical parameter (3-5); however, the assignment of the slow fraction to a distinct water population is difficult.Some investigators have suggested that the fast and slow water populations represent water from the extra-and intracellular spaces, respectively (6,7). The principal flaw in this assignment is that the fast and slow signal fractions are essentially the opposite of the known volume fractions for the two spaces. The fast component corresponds to approximately 70% of the detected signal, yet extracellular water comprises approximately 20% of total brain water. One possible explanation for this inconsistency is a difference in the T 2 relaxation properties of the two populations, but no TE dependence for signal fractions has been noted (1,8). A second flaw in this intra/extracellular assignment is the unproven assumption that all water in the intracellular space has a slow diffusion coefficient. In studies of large single cells, in which MRI can separate extra-and intracellular water signals spatially, some populations of intracellular water have been shown to have a relatively fast diffusion coefficient, especially water in the nucleus (9,10). Studies of diffusion of intracellular markers, such as 133 Cs ϩ , also suggest that intracellular water has a relatively fast diffusion coefficient (9). The assertion that extracellular water in tissues has a high ADC (fast diffusion) remains to be confirmed.We have tested the intra/extracellular assignment by evaluating intracellular water diffusion in the Xenopus oocyte. This well-characterized system (10) offers two advantages. The first is that the lifetime of the intracellular water preexchange lifetime is sufficiently long that measurements of intracellular water characteristics can be made without the complication of water exchange between the intra-and extracellular spaces. The second is that the characteristics of extracellular water can be easily manipulated with this sys...

The apparent diffusion coefficients (ADCs) of a series of markers concentrated in the extracellular space of normal rat brain were measured to evaluate, by inference, the ADC of water in the extracellular space. The markers (mannitol, phenylphosphonate, and polyethylene glycols) are defined as "compartment selective" because tissue culture experiments demonstrate some leakage into the intracellular space, making them less "compartment specific" than commonly believed. These primarily extracellular markers have ADCs similar to those of intracellular metabolites of comparable hydrodynamic radius, suggesting that water ADC values in the intra-and extracellular spaces are similar. If this is the case, then it is unlikely that a net shift of water from the extra-to the intracellular space contributes significantly to the reduction in water ADC detected following brain injury. Rather, this reduction is more likely due primarily to a reduction of the ADC of intracellular water associated with injury. Key words: diffusion; extracellular space; rat; stroke Diffusion-weighted imaging (DWI), in which contrast is based on the water apparent diffusion coefficient (ADC), is widely recognized as a useful tool for early detection of brain injury. Its utility in this setting arises from the observation that water ADC decreases rapidly following many forms of acute central nervous system (CNS) injury, including stroke (1), seizure (2), excitotoxic injury (3), trauma (4), hypoglycemia (5), and spreading depression (6). Despite the widespread clinical use of DWI, the pathophysiological mechanism(s) fundamental to changes in the water ADC remains poorly understood.Water motion in biological tissue is complex. The ADC is a quantification of incoherent molecular displacement which is sensitive to a wide range of incoherent microscopic motions, including passive Brownian thermal diffusion, active transport processes, and macroscopic motions which may include perivascular pulsation, cerebrospinal fluid (CSF) perfusion, and bulk brain motion. Further, water motion may be hindered or restricted by macromolecular binding, barriers such as cell membranes and the cytoskeletal matrix, and high protein concentrations. To complicate matters even more, the CNS water signal, and hence water ADC measurement, arises from molecules exchanging between the intracellular space (ICS) and the extracellular space (ECS), with water in each compartment possibly subject to different motions and barriers to motion. Following brain injury, active transport processes, intra-and extracellular volume fractions, intercompartment exchange rates, and other factors may change in association with an overall decrease in total water ADC. Due to the complex and unknown factors contributing to water motion in tissue, the descriptor "apparent" is applied to the diffusion coefficient determined by the pulsed-field-gradient method as the MR experimental rate constant characterizing this motion.The most frequently cited hypothesis explaining the reduction of brain water ADC f...

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. Key words: tetramethylammonium; tensor; intracellular; viscosityThe determinants of incoherent tissue-water displacement have been of increasing interest in recent years due to the clinical utility of diffusion-weighted MRI for the detection of brain injury. For example, the apparent diffusion coefficient (ADC) of water in mammalian brain decreases quickly following stroke (1), serving as an early indicator of injury. Numerous hypotheses have been put forward to explain this reduction in water ADC (1-3). One hypothesis posits that the overall decrease in water ADC is driven by a decrease in the ADC of intracellular water in response to injury. Evaluating this hypothesis requires compartment-specific measurements of water ADC values. This measurement has proven problematic in mammalian systems (but see the promising recent report by Silva et al. (4)). Herein we employ the Xenopus oocyte as a model system to explore displacement in the intracellular space. Intracellular displacement as measured by the NMR apparent diffusion experiment has been previously characterized in brain and cell systems for several molecules and ions (5-8), but the relationship between these ADCs and the water ADC is not obvious. The large size of the oocyte allows for direct measurement of ADC values of intracellular constituents, including water. Oocytes employed herein have volumes of 1.0 l with estimated intracellular aqueous volumes of 0.5 l.ADC reflects a measurement of incoherent water displacement over time that can be affected by many factors. One intriguing question about water displacement in the intracellular space regards the relative roles of passive, thermally driven (Brownian 1 ) diffusion vs. more active processes such as cytoplasmic streaming (10). One mechanism suggested for the decrease in intracellular water ADC described in the preceding paragraph is that the decrease is a consequence of energy failure and cessation of cytoplasmic streaming. Cell extracts from the Xenopus oocyte have previously been used as a model system in which to study some of the specific processes that can be more generally referred to as cytoplasmic streaming (11,12). In this study, the ADC values for water, as well as other molecules and ions of varying hydrodynamic ra...

Oocytes of Xenopus laevis are large, single cells that provide a promising model system for the exploration of the MR biophysics fundamental to more complex living systems. Previous studies have generally employed 2D spin-echo sequences with an image slice thickness greater than the thickness of the cellular volumes of interest. Also, the large cytoplasmic lipid signal has typically been ignored. This study describes separate, highresolution 3D measurements of the water and lipid spin densities, T 1 and T 2 relaxation time constants, and the water apparent diffusion rate constant (ADC) in the Xenopus oocyte without significant partial volume artifacts. The lipid spin-density and values for water MR properties varied monotonically from the vegetal to animal poles, indicating that the border between the poles is not sharply demarcated. The Xenopus laevis oocyte is a well-established cell model used in many branches of modern experimental biology. This versatile cell provides a promising model platform upon which to explore fundamental aspects of the MR biophysics underlying more complex living systems. In particular, the ability to spatially resolve the intracellular compartment allows the direct testing of hypotheses regarding various MR properties of the cytoplasm and nucleus. Recent efforts to integrate confocal microscopy and MR imaging will likely augment the capability of such experiments (1).Aguayo et al. (2) reported the first MR images of the Xenopus oocyte in 1986. Along with intriguing contrast between intracellular regions, the authors described a strong chemical shift artifact from cytoplasmic lipid. Posse and Aue (3) further characterized this lipid signal using a 4D spectroscopic imaging technique. Both studies localized the lipid signal to the cytoplasm, with little or no lipid signal arising from the nucleus. Since these initial studies, several investigators have calculated spin densities and T 2 and/or T 1 relaxation time constants for the oocyte nucleus, animal pole, and vegetal pole in control or altered cells (4 -6). The effect of the unknown, spatially varying lipid spin-density on these calculations, if any, has been largely ignored. Further, these studies have routinely employed 2D spin-echo sequences with slice profiles thicker than the cellular volumes of interest (VOIs), raising concerns regarding partial volume effects. This shortcoming is especially detrimental to studies of the nucleus, which has a diameter of only 300 m.Herein we present separate, high-resolution 3D measurements of the water and lipid spin densities, T 1 and T 2 relaxation time constants, and the water apparent diffusion coefficient (ADC) in the Xenopus oocyte without significant partial volume artifacts. We challenge the conventional binary segmentation of the oocyte cytoplasm into vegetal and animal poles and recognize cylindrical symmetry about the vegetal-animal (V-A) axis. Lipid-specific imaging is demonstrated for which water suppression is achieved via high diffusion weighting in the imaging sequence. We corr...

The exchange of water across biological membranes is of fundamental significance to both animal and plant physiology. Diffusional membrane permeability (P(d)) for the Xenopus oocyte, an important model system for water channel investigation, is typically calculated from intracellular water pre-exchange lifetime, cell volume, and cell surface area. There is debate, however, whether intracellular water motion affects water lifetime, and thereby P(d). Mathematical modeling of water transport is problematic because the intracellular water diffusion rate constant (D) for cells is usually unknown. The measured permeability may be referred to as the apparent diffusional permeability, P, to acknowledge this potential error. Herein, we show that magnetic resonance (MR) spectroscopy can be used to measure oocyte water exchange with greater temporal resolution and higher signal-to-noise ratio than other methods. MR imaging can be used to assess both oocyte geometry and intracellular water diffusion for the same single cells. MR imaging is used to confirm the dependence of intracellular water lifetime on intracellular diffusion. A model is presented to relate intracellular lifetime to true membrane diffusional permeability. True water diffusional permeability (2.7 +/- 0.4 microm/s) is shown to be 39 +/- 6% greater than apparent diffusional permeability for 8 oocytes. This discrepancy increases with cell size and permeability (such as after water channel expression) and decreases with increasing intracellular water D.