Chemically-fixed nervous tissues are well-suited for high-resolution, time-intensive MRI acquisitions without motion artifacts, such as those required for brain atlas projects, but the aldehyde fixatives used may significantly alter tissue MRI properties. To test this hypothesis, this study characterized the impact of common aldehyde fixatives on the MRI properties of a rat brain slice model. Rat cortical slices immersion-fixed in 4% formaldehyde demonstrated 21% and 81% reductions in tissue T 1 and T 2 , respectively (P < 0.001). The T 2 reduction was reversed by washing slices with phosphate-buffered saline (PBS) for 12 h to remove free formaldehyde solution. Diffusion MRI of cortical slices analyzed with a two-compartment analytical model of water diffusion demonstrated 88% and 30% increases in extracellular apparent diffusion coefficient (ADC EX ) and apparent restriction size, respectively, when slices were chemically-fixed with 4% formaldehyde (P ≤ 0.021). Further, fixation with 4% formaldehyde increased the transmembrane water exchange rate 239% (P < 0.001), indicating increased membrane permeability. Karnovsky's and 4% glutaraldehyde fixative solutions also changed the MRI properties of cortical slices, but significant differences were noted between the different fixative treatments (P < 0.05). The observed water relaxation and diffusion changes help better define the validity and limitations of using chemically-fixed nervous tissue for MRI investigations. Magn Reson Med 62:26 -34, 2009.
Ex vivo biological sample imaging can complement in vivo MRI studies. Since ex vivo studies are typically performed at room temperature, and samples are frequently preserved by fixation, it is important to understand how environmental and chemical changes dictated by ex vivo studies alter the physical and MR properties of a sample. Diffusion and relaxation time measurements were used to assess the effects of temperature change and aldehyde fixation on the biophysical and MR properties of a model biological tissue comprised of erythrocyte ghosts suspended in buffer or agarose gel. Sample temperature was varied between 10°C and 37°C. Diffusion MRI data were analyzed with a biophysically appropriate two-compartment exchange model. Temperature change resulted in a complex alteration of water diffusion properties due to the compartmental nature of tissues and alteration in membrane permeability. There has been a recent growth in the application of ex vivo MRI studies to biological tissues, organs, and entire organisms, which augment in vivo MRI by providing information that would be difficult or impossible to acquire from a living system. Ex vivo samples allow the use of stronger magnetic field gradients and permit a higher achievable signal-to-noise ratio (SNR) (also due to smaller RF coils and samples, and longer scan times) and thus higher spatial resolution, and produce data sets devoid of motion or flow artifacts. MRI of ex vivo samples has been used for morphological evaluations of human samples, particularly in neurological (1-4) and cardiac studies (5-7). A variety of animal models have been used in a similar fashion (8,9). For example, MR microscopy of fixed mice and mouse organs has been employed for morphological phenotyping (10,11), and has been widely used in diffusion tensor imaging (DTI) studies investigating the microstructure of the central nervous system (e.g., Refs. 12 and 13). Aldehyde fixation is frequently employed to preserve tissues from degradation prior to imaging, and MR data are most commonly acquired at room temperature (approximately 20°C) rather than physiological temperature (ϳ37°C). However, it is recognized that the process of fixation may alter the morphological and physical properties of the fixed tissue, as well as the MR properties, and that these will vary with the fixative and fixation technique used (4,6,14,15). To relate data acquired from chemically fixed samples at room temperature to an in vivo situation, it is necessary to understand how these environmental and sample changes will affect the MR-visible properties of the sample. We hypothesized that diffusion MRI could be used to detect and quantify changes in tissue biophysical properties (such as membrane permeability and water diffusion rates) and MR properties (such as relaxation rates) caused by aldehyde fixation and/or changes in temperature.The effects of fixation on biophysical and water properties of the model tissue were investigated for three fixative types. Fixatives such as formaldehyde and glutaraldehyde cros...
High-resolution imaging of human autopsy tissues may improve our understanding of in vivo MRI findings, but interpretation is complicated because samples are obtained by immersion fixation following a postmortem interval (PMI). This study tested the hypotheses that immersion fixation and PMI's from 0 - 24 hours would alter the water relaxation and diffusion properties in rat cortical slice and spinal cord models of human nervous tissue. Diffusion data collected from rat cortical slices at multiple diffusion times (10 - 60 ms) and b-values (7 - 15,000 s/mm2) were analyzed using a two-compartment model with exchange. Rat spinal cords were characterized with standard diffusion tensor imaging (21 directions, b = 1250 s/mm2). Switching from perfusion- to immersion-fixation at 0-hrs PMI altered most MRI properties of rat cortical slices and spinal cords, including a 22% decrease in fractional anisotropy (P < 0.001). After 4 hrs PMI, cortical slice T1 and T2 increased 22% and 65% respectively (P < 0.001), transmembrane water exchange decreased 23% (P < 0.001) and intracellular proton fraction increased 25% (P = 0.002). After 6 hrs PMI, spinal cord white matter fractional anisotropy had decreased 38% (P < 0.001). MRI property changes were observed for PMIs up to 24 hours. The MRI changes correlated with protease activity and histopathological signs of autolysis. Thus, immersion fixation and/or even short PMIs (4-6 hours) altered the MRI properties of rat nervous tissue. This suggests comparisons between in vivo clinical MRI and MRI data from human autopsy tissues should be interpreted with caution.
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