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On-line visualization of dye diffusion in fresh unfixed human skin.

PURPOSE: The purpose of the current study was to develop a new method to examine the diffusion in fresh unfixed human skin on-line. METHODS: Full thickness skin samples were cut perpendicular to the skin surface (cutting plane facing upwards) with a new cutting device forming part of the final diffusion cell. The donor solution contained 0.1 mg/ml Bodipy FL C5 (moderately lipophilic) dissolved in citric acid buffer, pH 5.0, and the acceptor phase consisted of phosphate-buffered saline, pH 7.4. Images were taken with confocal laser scanning microscopy (CLSM) every 10 min for 8 h. RESULTS: This new method enabled for the first time visualization of concentration profiles in different skin layers simultaneously as a function of time. For this model penetrant, Bodipy FL C5 showed that the lower stratum corneum layer constitutes the greatest barrier to diffusion. Furthermore, there is preferred partitioning of this probe in epidermis vs. either stratum corneum or dermis. CONCLUSIONS: The on-line diffusion cell in combination with CLSM is a promising tool to study diffusion processes of dyes in fresh unfixed skin on-line. The method has the potential to access deeper skin layers as well as to visualize diffusion processes in cells.

Algorithms↗

Isoflurane strongly affects the diffusion of intracellular metabolites, as shown by 1H nuclear magnetic resonance spectroscopy of the monkey brain.

Isoflurane is a volatile anesthetic commonly used for animal studies. In particular, diffusion nuclear magnetic resonance (NMR) spectroscopy is frequently performed under isoflurane anesthesia. However, isoflurane is known to affect the phase transition of lipid bilayer, possibly resulting in increased permeability to metabolites. Resulting decreased restriction may affect metabolite apparent diffusion coefficient (ADC). In the present work, the effect of isoflurane dose on metabolite ADC is evaluated using diffusion tensor spectroscopy in the monkey brain. For the five detected intracellular metabolites, the ADC exhibits a significant increase when isoflurane dose varies from 1% to 2%: 13%+/-8% for myo-inositol, 14%+/-13% for total N-acetyl-aspartate, 20%+/-18% for glutamate, 27%+/-7% for total creatine and 53%+/-17% for total choline. Detailed analysis of ADC changes experienced by the five different metabolites argues in favor of facilitated metabolite exchange between subcellular structures at high isoflurane dose. This work strongly supports the idea of metabolite diffusion in vivo being significantly restricted in subcellular structures at long diffusion time, and provides new insights for interpreting ADC values as measured by diffusion NMR spectroscopy.

Anesthetics, Inhalation↗

Quantitative metrics for evaluating parallel acquisition techniques in diffusion tensor imaging at 3 Tesla.

OBJECTIVES: Single-shot echo-planar based diffusion tensor imaging is prone to geometric and intensity distortions. Parallel imaging is a means of reducing these distortions while preserving spatial resolution. A quantitative comparison at 3 T of parallel imaging for diffusion tensor images (DTI) using k-space (generalized auto-calibrating partially parallel acquisitions; GRAPPA) and image domain (sensitivity encoding; SENSE) reconstructions at different acceleration factors, R, is reported here. MATERIALS AND METHODS: Images were evaluated using 8 human subjects with repeated scans for 2 subjects to estimate reproducibility. Mutual information (MI) was used to assess the global changes in geometric distortions. The effects of parallel imaging techniques on random noise and reconstruction artifacts were evaluated by placing 26 regions of interest and computing the standard deviation of apparent diffusion coefficient and fractional anisotropy along with the error of fitting the data to the diffusion model (residual error). RESULTS: The larger positive values in mutual information index with increasing R values confirmed the anticipated decrease in distortions. Further, the MI index of GRAPPA sequences for a given R factor was larger than the corresponding mSENSE images. The residual error was lowest in the images acquired without parallel imaging and among the parallel reconstruction methods, the R = 2 acquisitions had the least error. The standard deviation, accuracy, and reproducibility of the apparent diffusion coefficient and fractional anisotropy in homogenous tissue regions showed that GRAPPA acquired with R = 2 had the least amount of systematic and random noise and of these, significant differences with mSENSE, R = 2 were found only for the fractional anisotropy index. CONCLUSION: Evaluation of the current implementation of parallel reconstruction algorithms identified GRAPPA acquired with R = 2 as optimal for diffusion tensor imaging.

Adult↗

Elevated CNS average diffusion constant in Fabry disease.

AIMS: Evaluation of the average brain diffusion constant in Fabry disease. INTRODUCTION: Fabry disease is an X-linked recessive lysosomal storage disorder secondary to deficiency of alpha-galactosidase A and resulting in excess tissue globotriaosylceramide, particularly in cerebral blood vessels. This has been associated with cerebral hyperperfusion. Increased tissue perfusion should increase interstitial water by the Starling relationship. This hypothesis was examined by measuring the average CNS diffusion constant (Dav) in patients with Fabry disease using diffusion-weighted magnetic resonance imaging (DWI). METHODS: Axial DWI was performed at b=0 seconds/mm2 and b = 1000 seconds/mm2 (TR (pulse repetition time), 10000; TE (time to echo), 100; FOV (field of view), 22 cm: 3 mm interleaved slices; image matrix, 128 x 128; GE Signa, 1.5T). Eight healthy male volunteers (age range, 21-47 years) and 17 hemizygous patients with Fabry disease (age range, 19-49 years) were examined. Following DWI acquisition, the trace image and the diffusion distribution map were calculated. The diffusion distribution curve was then fitted by a multi-modal Gaussian curve, allowing estimation of Dav. RESULTS: The Dav was 0.743 +/- 0.024 x 10(-5) cm2/second (mean +/- SD) for patients with Fabry disease and 0.726 +/- 0.014 x 10(-5) cm2/second for the control group. Dav was significantly increased in the patients with Fabry disease compared with the controls (p = 0.029) CONCLUSIONS: The elevated Dav indicates increased brain tissue water diffusivity in patients with Fabry disease, a finding consistent with increased extracellular water and increased cerebral blood flow.

Adult↗

Role of molecular diffusion in conventional and high frequency ventilation.

The influence of molecular diffusion on gas-mixing during conventional mechanical ventilation (CMV) and high frequency ventilation (HFV) was studied by observing the wash-in of six poorly soluble, inert gases in arterial blood. Anesthetized dogs were ventilated either with CMV or HFV. Following a step change in inspired gas composition, the increase in arterial concentrations of hydrogen, helium, methane, ethane, isobutane, and sulfur hexafluoride was determined by gas chromatography. The relative gas diffusivities encompassed a range of almost one order of magnitude. Propane, present in inspired gas during both the control and wash-in phases, served as an internal reference for calculation of blood tracer concentrations. The wash-in of all six inert gases followed a single exponential time course during both CMV and HFV. The rate of wash-in of each gas decreased with increasing molecular weight (MW). The relationship of rate constants to a measure of relative diffusivity (MW-0.5) was significantly different than zero for both types of ventilation. The slope of this relationship was three times larger for CMV than HFV, indicating that molecular diffusion has a greater role in gas mixing during ventilation with large tidal volumes. Diffusion has a minor role in gas mixing during high frequency ventilation with small tidal volumes. Demonstration of the presence of gas separation secondary to molecular diffusion during HFV is enhanced by measuring wash-in, rather than wash-out, of inert gases because gas separation is likely to be obscured as exhaled gases pass through the well-mixed central airways during gas wash-out.

Animals↗

Evaluation of diffusion in gel entrapment cell culture within hollow fibers.

AIM: To investigate diffusion in mammalian cell culture by gel entrapment within hollow fibers. METHODS: Freshly isolated rat hepatocytes or human oral epidermoid carcinoma (KB) cells were entrapped in type I collagen solutions and statically cultured inside microporous and ultrafiltration hollow fibers. During the culture time collagen gel contraction, cell viability and specific function were assessed. Effective diffusion coefficients of glucose in cell-matrix gels were determined by lag time analysis in a diffusion cell. RESULTS: Significant gel contractions occurred in the collagen gels by entrapment of either viable hepatocytes or KB cells. And the gel contraction caused a significant reduction on effective diffusion coefficient of glucose. The cell viability assay of both hepatocytes and KB cells statically cultured in hollow fibers by collagen entrapment further confirmed the existence of the inhibited mass transfer by diffusion. Urea was secreted about 50% more by hepatocytes entrapped in hollow fibers with pore size of 0.1 mum than that in hollow fibers with MWCO of 100 ku. CONCLUSION: Cell-matrix gel and membrane pore size are the two factors relevant to the limited mass transfer by diffusion in such gel entrapment of mammalian cell culture.

Animals↗

A quantitative study of water diffusion in multiple sclerosis lesions and normal-appearing white matter using echo-planar imaging.

OBJECTIVES: We used an optimized echo-planar pulse sequence for isotropically weighted diffusion imaging (1) to measure mean diffusivity (D) in lesions and normal-appearing white matter (NAWM) in the entire brain from patients with mildly disabling relapsing-remitting multiple sclerosis (MS), (2) to compare the D of NAWM from patients with that of white matter from normal controls, (3) to evaluate whether lesions classified on the basis of their appearance on enhanced T1-weighted scans have different D, and (4) to investigate the relationship between diffusion changes in lesions and NAWM. METHODS: Dual-echo and diffusion-weighted scans were obtained from 35 patients with relapsing-remitting MS and 24 sex- and age-matched normal controls. Postcontrast T1-weighted images were also obtained from the patients. After creating D maps and image coregistration, D values were measured for MS lesions larger than 5 mm and for 22 NAWM areas from each subject. RESULTS: All NAWM areas studied had significantly higher D in patients than in controls. A total of 173 lesions were identified on the dual-echo scans from patients. The average D for these lesions was significantly higher than that of NAWM. Twenty-two lesions were enhancing and 60 were classified as T1-hypointense. No significant difference in D values was found between enhancing and nonenhancing lesions, while the average D of T1-hypointense lesions was significantly higher than the average D of T1-isointense lesions. There was no significant correlation between the average D in lesions and NAWM. CONCLUSIONS: This study shows that diffusion-weighted imaging is able to identify MS lesions with severe tissue disruption. It also shows that widespread increased diffusion can be measured in the NAWM from patients with MS, and suggests that such changes are, at least partially, independent of larger abnormalities.

Adult↗

Diffusion-tensor magnetic resonance imaging detects normal-appearing white matter damage unrelated to short-term disease activity in patients at the earliest clinical stage of multiple sclerosis.

BACKGROUND: Diffusion-tensor (DT) magnetic resonance imaging (MRI) has the potential to elucidate some characteristics of tissue microstructure inaccessible to other MRI techniques. OBJECTIVE: To investigate whether normal-appearing brain tissue abnormalities occur in patients with multiple sclerosis at the earliest clinical stage and whether their severity is predictive of a short-term disease evolution by using DT MRI. DESIGN: Forty-five patients and 22 healthy control subjects were studied. All patients had had a clinically isolated syndrome within the 3 months preceding study enrollment and paraclinical evidence of disease dissemination in space. During a single session, dual-echo, pulsed-gradient spin-echo echo-planar, and postgadolinium T1-weighted images of the brain were obtained from each subject. In patients, dual-echo and enhanced images were obtained after 3 and 12 months, to detect MRI signs of disease dissemination in time. An on-study neurological examination was also conducted to ascertain the occurrence of clinical relapses. Mean diffusivity and fractional anisotropy maps were derived from DT images. Normal-appearing white matter (NAWM) and normal-appearing gray matter mean diffusivity and fractional anisotropy histograms were produced and analyzed. RESULTS: During the study period, 29 patients showed MRI evidence of disease dissemination in time. When compared with healthy controls, patients showed higher average NAWM mean diffusivity (P = .01), lower average NAWM mean diffusivity peak height (P < .001), and fractional anisotropy (P < .001). The DT MRI characteristics of patients did not differ between those with and those without disease dissemination in time at follow-up. CONCLUSIONS: In patients with multiple sclerosis at the earliest clinical stage, the severity of NAWM damage does not predict new lesion formation in the short term, suggesting that the "diffuse" component of tissue damage is, at least partially, independent of the "discrete," predominantly inflammatory aspects of the disease since its clinical onset.

Adult↗

Measurement of Gd-DTPA diffusion through PVA hydrogel using a novel magnetic resonance imaging method.

Polyvinyl alcohol-cryogel (PVA-C) is a hydrogel that is an excellent tissue mimic. In order to characterize mass transfer in this material, as well as to demonstrate in principle the ability to noninvasively measure solute diffusion in tissue, we measured the diffusion coefficient of the magnetic resonance (MR) contrast agent gadolinium diethylene triaminopentaacetic acid (Gd-DTPA) through PVA-C using a clinical MR imager. The method involved filling thick-walled rectangular PVA-C "cups" with known concentrations of Gd-DTPA solutions. Then by using a fast inversion recovery spin echo MR imaging protocol, a signal "null" contour was created in the MR image that corresponded to a second, known concentration of Gd-DTPA. By collecting a series of MR images through the PVA-C wall as a function of time, the displacement of this second known isoconcentration contour could be tracked. Application of Fick's second law of diffusion yielded the diffusion coefficient. Seven separate experiments were performed using various combinations of initial concentrations of Gd-DTPA within the PVA-C cups (3.2, 25.6, or 125 mM) and tracked isoconcentrations contours (0.096, 0.182, or 0.435 mM Gd-DTPA). The experimental results and the predictions of Fick's law were in excellent agreement. The diffusivity of Gd-DTPA through 10% PVA hydrogel was found to be (2.6 +/- 0.04) x 10(-10) m(2)/s (mean +/- s.e.m.). Separate permeability studies showed that the diffusion coefficient of Gd-DTPA through this hydrogel did not change with an applied pressure of up to 7.1 kPa. Accurate measurements could be made within 30 min if suitable Gd-DTPA concentrations were selected. Due to the excellent repeatability and fast data acquisition time, this technique is very promising for future in vivo studies of species transport in tissue.

Calibration↗

Changes in lipid diffusibility in the hamster sperm head plasma membrane during capacitation in vivo and in vitro.

The technique of fluorescence recovery after photobleaching (FRAP) was employed on spermatozoa labeled with the fluorescent lipid analogue C14dil to provide two measures of lateral diffusion in the plane of the sperm plasma membrane during capacitation in vivo and in vitro: the diffusion coefficient (D) for C14dil and the fraction of C14dil that is free to diffuse (%R) within the domain. To evaluate changes in lipid diffusibility during capacitation in vivo, spermatozoa were recovered from the uterus within 30 min after ejaculation or from the oviduct at 2, 4, 6 and 8 hr after mating. To compare the changes which occur in vivo with those which occur during capacitation in vitro, caudal epididymal spermatozoa were incubated under capacitating or non-capacitating (control) conditions for 4 hr. Although transient changes in D occurred during the course of capacitation, there was no net change in D for either anterior (AH) or posterior head (PH) domains following capacitation in vitro or in vivo. Significant differences in the lipid diffusion coefficient between the two head domains were observed during the course of capacitation. A transient decrease in %R was observed for the AH domain during capacitation in vitro and incubation under control conditions, but no significant change in %R was observed in the AH domain during capacitation in vivo. A significant decline in %R of the PH domain was observed for spermatozoa during capacitation in vivo, in vitro and following incubation under non-capacitating conditions. These data indicate that the changes in the lipid diffusibility of the AH and PH domains which occur during capacitation in vivo exhibit both similarities and differences to those which occur during capacitation in vitro.

Animals↗

Regional and directional anisotropy of apparent diffusion coefficient in rat brain.

Quantitative diffusion maps were recorded in normal rat brain. In multi-slice sections covering the whole brain, strong variation of the apparent diffusion coefficient (ADC) was observed depending on slice position at constant gradient direction. Furthermore, a varying difference between apparent diffusion coefficients depending on gradient direction was found, reaching 32% in the cortex of the ventral-most horizontal sections while showing equal ADC on the dorsal cortex side. The regional variation and directional anisotropy of the ADC was not restricted to white matter but was described for both cortical and subcortical brain tissue. From diffusion coefficients along the three major field gradient directions (ADCx, ADCy, ADCz), the average ADC (ADCaverage) was determined from the trace of the diffusion tensor (D) as 653+/-28 microm2/s for parietal cortex and 671+/-32 microm2/s for lateral cortex, independent of position along the sagittal direction. From these observations about the regional diffusion anisotropy, a more stringent protocol for the description of ischemic ADC changes is proposed.

Animals↗

Self-diffusion of water in cartilage and cartilage components as studied by pulsed field gradient NMR.

Pulsed field gradient (PFG) nuclear magnetic resonance (NMR) was used to investigate the self-diffusion behavior of water molecules in cartilage, polymeric cartilage components, and different model polymers. The short-time self-diffusion coefficients (diffusion time delta approximately/= 13 msec) are found to decrease steadily with decreasing water content. This holds equally well for cartilage and cartilage components. The short-time diffusion coefficients are subjected to a rather nonspecific obstruction effect and mainly depend on the water content of the sample. The long-time diffusion coefficients in cartilage (delta approximately/= 500 msec), however, reflect structural properties of this tissue. Measurements with varying observation times as well as experiments involving enzymatic treatment of articular cartilage suggest that the collagenous network in cartilage is likely to be responsible for the observed restricted diffusion.

Animals↗

The coexistence of nodular and diffuse patterns in nodular non-Hodgkin's lymphomas: significance and clinicopathologic correlation.

One hundred and twenty-six cases of nodular lymphoma have been classified cytologically by the criteria of Rappaport, and have been divided into three architectural groups, based on the degree of nodularity. Eighty-five percent of the patients have been followed for at least 5 years after initial therapy. Analysis of actuarial survival curves reveals that patients in the poorly differentiated lymphocytic and mixed histiocytic-lymphocytic categories with the three architectural patterns ("nodular only," "nodular with focal diffuse," and "nodular with diffuse") have similar long-term and disease-free survivals. Nevertheless, any degree of nodularity imparts a more favorable prognosis than diffuse lymphoma of coreresponding cell type. In the small number of patients with a nodular lye than in the other two histiocytic type, associated with diffuse areas, the prognosis is less favorable than in the other two histologic groups. Thus, a nodular lymphoma of so-called histiocytic type with diffuse areas may behave more like a diffuse than nodular lymphoma, and warrants appropriate therapy.

Female↗

Measuring the translational diffusion coefficients of small DNA molecules by capillary electrophoresis.

The apparent translational diffusion coefficients of four 20 base pair (bp) DNA oligonucleotides with different sequences have been measured by capillary electrophoresis, using the stopped migration method. The diffusion coefficients of the four oligomers were equal within experimental error, and averaged (120 +/- 10) x 10(-8) cm(2) s(-1) in 40 mM Tris-acetate-EDTA buffer at 25 degrees C. Since this value is nearly identical to the translational diffusion coefficient determined for a different 20-bp oligomer using other methods, the stopped migration method can accurately measure the diffusion coefficients of small DNA oligomers. The apparent diffusion coefficient of a 118-bp DNA restriction fragment was also measured by the stopped migration method. However, the observed value was approximately 25% larger than expected from other measurements, possibly because the diffusion coefficients of larger DNA molecules are somewhat dependent on the ionic strength of the solution.

DNA↗

Diffusion tensor MRI of the spinal cord.

Apparent diffusion tensor (ADT) measurements on the spinal cord using a pulsed-field-gradient (PFG) multi-shot echo-planar imaging (EPI) sequence are presented. In a study of 10 healthy volunteers, the obtained rotationally invariant anisotropy information is compared to the results obtained by the rotationally dependent methods. The water diffusivity in the direction parallel to the fibers was found to be almost 2.5 times higher than the average diffusivity in directions perpendicular to the fibers and showed cylindrically symmetric anisotropy characteristics. The influence of partial volume effects and the point spread function on the measured results was evaluated, and it was the concluded that a resolution of 1 mm in the read and phase directions is required to obtain unbiased values. Possible clinical implications were demonstrated by investigating the diffusion characteristics of 10 patients suffering from narrowing of the cervical canal. The changes in the diffusion characteristics were found to be large enough to allow a robust detection of diffusion changes in the spine, even in cases in which conventional T(2) and T(1) weighted images were unable to detect any lesion.

Anisotropy↗

Apparent diffusion tensor measurements in myelin-deficient rat spinal cords.

The apparent diffusion tensor (ADT) was measured in excised and fixed spinal cords from myelin-deficient (md) rats and age-matched controls. These data were used to obtain the principal diffusivities of the ADT, and also the scalar invariant parameters _D (averaged principal diffusivity) and A(sigma) (anisotropy index) for four white matter and two gray matter regions. The results for white matter regions showed that the principal diffusivities were significantly higher for md animals, and while the _D was increased in tissue from md animals, the A(sigma) was found to be decreased. Grey matter _D was measured to be between those of white matter from control and md animals, and the A(sigma) was much smaller than that of white matter from both sets of animals, indicating that diffusion in md white matter is more anisotropic than in gray matter. The results show that while myelination is not a prerequisite for diffusion anisotropy, it does influence the magnitude of the observed anisotropy. Magn Reson Med 45:191-195, 2001.

Animals↗

Diffusion, Joule heating, and band broadening in capillary gel electrophoresis of DNA.

We calculated the longitudinal and transverse diffusion coefficients for a DNA molecule undergoing gel electrophoresis in the limit where it is reptating through a dense polymer matrix. Our results indicate that both diffusion coefficients increase with the electric field intensity. The transverse and longitudinal diffusion coefficients are roughly equal for regular field intensities, but the former dominates for the high field intensities normally used for capillary gel electrophoresis (CE). This has important implications for the optimization of CE. Our results clearly show that the naive use of the zero-field diffusion constant, the Einstein relation or the longitudinal diffusion constant when calculating the contribution of the parabolic temperature profile to band broadening may lead to large overestimates under typical CE conditions. Finally, we show that the field-dependent diffusion coefficients may be responsible for the existence of an optimal field intensity for CE, even if Joule heating is neglected.

DNA↗

Formal characterization and extension of the linearized diffusion tensor model.

We analyzed the properties of the logarithm of the Rician distribution leading to a full characterization of the probability law of the errors in the linearized diffusion tensor model. An almost complete lack of bias, a simple relation between the variance and the signal-to-noise ratio in the original complex data, and a close approximation to normality facilitated estimation of the tensor components by an iterative weighted least squares algorithm. The theory of the linear model has also been used to derive the distribution of mean diffusivity, to develop an informative statistical test for relative lack of fit of the ellipsoidal (or spherical) model compared to an unrestricted linear model in which no specific shape is assumed for the diffusion process, and to estimate the signal-to-noise ratios in the original data. The false discovery rate (FDR) has been used to control thresholds for statistical significance in the context of multiple comparisons at voxel level. The methods are illustrated by application to three diffusion tensor imaging (DTI) datasets of clinical interest: a healthy volunteer, a patient with acute brain injury, and a patient with hydrocephalus. Interestingly, some salient features, such as a region normally comprising the basal ganglia and internal capsule, and areas of edema in patients with brain injury and hydrocephalus, had patterns of error largely independent from their mean diffusivities. These observations were made in brain regions with sufficiently large signal-to-noise ratios (>2) to justify the assumptions of the log Rician probability model. The combination of diffusivity and its error may provide added value in diagnostic DTI of acute pathologic expansion of the extracellular fluid compartment in brain parenchymal tissue.

Adult↗