Wolfgang Dreher

Restricted diffusion and exchange of intracellular water: theoretical modelling and diffusion time dependence of 1H NMR measurements on perfused glial cells

Intracellular diffusion properties of water in F98 glioma cells immobilized in basement membrane gel threads, are investigated with a pulsed‐field‐gradient spin‐echo NMR technique at diffusion times from 6 to 2000 ms and at different temperatures. In extended model calculations the concept of ‘restricted intracellular diffusion at permeable boundaries’ is described by a combined Tanner–Kärger formula. Signal components in a series of ct experiments (constant diffusion time) are separated due to different diffusion properties (Gaussian and restricted diffusion), and physiological as well as morphological cell parameters are extracted from the experimental data. The intracellular apparent diffusion coefficients strongly depend on the diffusion time and are up to two orders of magnitude smaller than the self diffusion constant of water. Propagation lengths are found to be in the range of 4–7 μm. Hereby intracellular signals of compartments with a characteristic diameter could be selected by an appropriate gradient strength. With cg experiments (constant gradient) a mean intracellular residence time for water is determined to be about 50 ms, and the intrinsic intracellular diffusion constant is estimated to 1 × 10−3  mm2 /s. Studying the water diffusion in glial cells provides basic understanding of the intracellular situation in brain tissue and may elucidate possible influences on the changes in the diffusion contrast during ischemic conditions.

DETECTION OF HOMONUCLEAR DECOUPLED IN VIVO PROTON NMR SPECTRA USING CONSTANT TIME CHEMICAL SHIFT ENCODING: CT-PRESS

A new pulse sequence, termed CT-PRESS, is presented, which allows the detection of in vivo 1H NMR spectra with effective homonuclear decoupling. A PRESS sequence with a short echo-time TE, used for spatial localization, is supplemented by an additional 180 degrees pulse. The temporal position of this 180 degree pulse is shifted within a series of experiments, while the time interval between signal excitation and detection is kept constant. CT-PRESS is a two-dimensional (2D) spectroscopic experiment as far as data acquisition and processing are concerned, although only diagonal signals are generated in the 2D spectrum. However, since the principle of constant time chemical shift encoding is used in the t1 domain, effective homonuclear decoupling is obtained by projecting the 2D spectrum onto the corresponding f1 axis. Thus, good spectral resolution and high signal-to-noise ratio are obtained. The main advantage, as compared to localized 2D J-resolved MRS, is that optimized experiments can be performed for coupled resonances of interest by choosing the sequence parameters dependent on the type of multiplets, the J-coupling constants and T2. Major fields of application will be parametric studies on coupled resonances, (e.g., T1, diffusion behavior or magnetization transfer) and/or the detection of spatial and temporal changes of metabolites with coupled spin systes.

Changes in apparent diffusion coefficients of metabolites in rat brain after middle cerebral artery occlusion measured by proton magnetic resonance spectroscopy.

Diffusion‐weighted proton MR spectroscopy and imaging have been applied to a rat brain model of unilateral middle cerebral artery occlusion between 1 and 4 hr post occlusion. Similar apparent diffusion coefficients (ADC) of most metabolites were observed within each hemisphere. In the ischemic ipsilateral hemisphere, the ADCs were (0.083–0.116) · 10–3 mm2/sec for lactate (Lac), alanine (Ala), γ‐amino butyric acid (GABA), N‐acetyl aspartate (NAA), glutamine (Gln), glutamate (Glu), total creatine (tCr), choline‐containing compounds (Cho), and myo‐inositol (Ins), in the contralateral hemisphere (0.138–0.158) · 10–3 mm2/sec for NAA, Glu, tCr, Cho, and Ins. Higher ADCs was determined for taurine (Tau) in the ipsilateral (0.144 · 10–3 mm2/sec) and contralateral (0.198 · 10–3 mm2/sec) hemisphere. In the ischemic hemisphere, a relative ADC decrease to 65–75% was observed for NAA, Glu, tCr, Cho, Ins and Tau, which was similar to the decrease of the water ADC (to 67%). The results suggest a common cause of the observed ADC changes and provide a broader experimental basis to evaluate theories of water and metabolite diffusion. Magn Reson Med 45:383–389, 2001.

Diffusion in compartmental systems. I. A comparison of an analytical model with simulations

This article examines the way in which microscopic tissue parameters affect the signal attenuation of diffusion‐weighted MR experiments. The influence of transmembrane water flux on the signal decay is emphasized using the Kärger equations, which are modified with respect to the cellular boundary restrictions for intra‐ and extracellular diffusion. This analytical approach is extensively compared to Monte‐Carlo simulations for a tissue model consisting of two compartments. It is shown that diffusion‐weighted MR methods provide a unique tool for estimation of the intracellular exchange time. Restrictions of applicability to in vivo data are examined. It is shown that the intracellular exchange time strongly depends on the size of a cell, leading to an apparent diffusion time dependence for in vivo data. Hence, an analytical model of a two‐compartment system with an averaged exchange time is inadequate for the interpretation of signal curves measured in vivo over large ranges of b‐values. Furthermore, differences of multiexponential signal curves, as obtained by different methods of diffusion weighting, can be explained by the influence of transmembrane water flux. Magn Reson Med 50:500–509, 2003.

BLBP-expression in astrocytes during experimental demyelination and in human multiple sclerosis lesions

Several lines of evidence indicate that remyelination represents one of the most effective mechanisms to achieve axonal protection. For reasons that are not yet understood, this process is often incomplete or fails in multiple sclerosis (MS). Activated astrocytes appear to be able to boost or inhibit endogenous repair processes. A better understanding of remyelination in MS and possible reasons for its failure is needed. Using the well-established toxic demyelination cuprizone model, we created lesions with either robust or impaired endogenous remyelination capacity. Lesions were analyzed for mRNA expression levels by Affymetrix GeneChip® arrays. One finding was the predominance of immune and stress response factors in the group of genes which were classified as remyelination-supporting factors. We further demonstrate that lesions with impaired remyelination capacity show weak expression of the radial-glia cell marker brain lipid binding protein (BLBP, also called B-FABP or FABP7). The expression of BLBP in activated astrocytes correlates with the presence of oligodendrocyte progenitor cells. BLBP-expressing astrocytes are also detected in experimental autoimmune encephalomyelitis during the remission phase. Furthermore, highest numbers of BLBP-expressing astrocytes were evident in lesions of early MS, whereas significantly less are present at the rim of (chronic)-active lesions from patients with long disease duration. Transfection experiments show that BLBP regulates growth factor expression in U87 astrocytoma cells. In conclusion, we provide evidence that expression of BLBP in activated astrocytes negatively correlates with disease duration and in parallel with remyelination failure.

Diffusion in compartmental systems. II. Diffusion‐weighted measurements of rat brain tissue in vivo and postmortem at very large b‐values

Diffusion‐weighted single‐voxel 1H spectroscopic measurements were performed on rat brain tissue in vivo and postmortem. Diffusion weighting was achieved by varying the diffusion time from 23 ms to 1.18 sec via the mixing time in a stimulated echo sequence. A series of constant gradient (cg‐) experiments of eight effective gradient strengths q̃2(q̃2 = γ2δ2g2) from 24.2 × 103 to 490.2 × 103 mm−2 was performed, resulting in a maximum attenuation factor of b = 580,000 s/mm2. A fit of three exponential terms was found to be appropriate to represent the attenuation signal over the whole b‐range. The behavior of the slowest decaying component can be fully understood in terms of a long time limit of a modified Kärger formalism for a two‐compartment system. This allowed estimation of the transmembrane water exchange rate: the intracellular exchange time was determined to be 622 ± 29 ms and 578 ± 20 ms in vivo and postmortem, respectively. Magn Reson Med 50:510–514, 2003.

New method for the simultaneous detection of metabolites and water in localized in vivo 1H nuclear magnetic resonance spectroscopy.

A new two‐scan method for localized 1H in vivo NMR spectroscopy (MRS) without water suppression (WS) is described. In one of the scans, two chemical shift selective 180° pulses are applied prior to a standard localization sequence to invert all metabolite signals upfield and downfield from water, which remains unaffected. The difference spectrum records the metabolites whereas water and accompanying gradient induced artifacts are widely suppressed. The method was implemented on a 4.7‐T system using point resolved spectroscopy with a short echo time of 18 ms. Phantom measurements proved the feasibility of absolute quantification using water as an internal reference. Measurements on healthy rat brain yielded comparable spectrum quality as measurements with water presaturation. The method does not require additional adjustments or sophisticated data postprocessing and scales favorably with increasing B0 field. Therefore, the method should be useful for 1H MRS without WS. Although the two‐step method doubles the minimum total measurement time, it may also be of interest for spectroscopic imaging (SI) without WS, in particular if fast SI techniques are applied. Magn Reson Med 54:190–195, 2005.

Toward quantitative short‐echo‐time in vivo proton MR spectroscopy without water suppression

A methodological development for quantitative short‐echo‐time (TE) in vivo proton MR spectroscopy (MRS) without water suppression (WS) is described that integrates experimental and software approaches. Experimental approaches were used to eliminate frequency modulation sidebands and first‐order phase errors. The dominant water signal was modeled and extracted by the matrix pencil method (MPM) and was used as an internal reference for absolute metabolite quantification. Spectral fitting was performed by combining the baseline characterization by a wavelet transform (WT)‐based technique and time‐domain (TD) parametric spectral analysis using full prior knowledge of the metabolite model spectra. The model spectra were obtained by spectral simulation instead of in vitro measurements. The performance of the methodology was evaluated by Monte Carlo (MC) studies, phantom measurements, and in vivo measurements on rat brains. More than 10 metabolites were quantified from spectra measured at TE = 20 ms on a 4.7 T system. Magn Reson Med, 2006.

2D and 3D MALDI-imaging: conceptual strategies for visualization and data mining.

3D imaging has a significant impact on many challenges in life sciences, because biology is a 3-dimensional phenomenon. Current 3D imaging-technologies (various types MRI, PET, SPECT) are labeled, i.e. they trace the localization of a specific compound in the body. In contrast, 3D MALDI mass spectrometry-imaging (MALDI-MSI) is a label-free method imaging the spatial distribution of molecular compounds. It complements 3D imaging labeled methods, immunohistochemistry, and genetics-based methods. However, 3D MALDI-MSI cannot tap its full potential due to the lack of statistical methods for analysis and interpretation of large and complex 3D datasets. To overcome this, we established a complete and robust 3D MALDI-MSI pipeline combined with efficient computational data analysis methods for 3D edge preserving image denoising, 3D spatial segmentation as well as finding colocalized m/z values, which will be reviewed here in detail. Furthermore, we explain, why the integration and correlation of the MALDI imaging data with other imaging modalities allows to enhance the interpretation of the molecular data and provides visualization of molecular patterns that may otherwise not be apparent. Therefore, a 3D data acquisition workflow is described generating a set of 3 different dimensional images representing the same anatomies. First, an in-vitro MRI measurement is performed which results in a three-dimensional image modality representing the 3D structure of the measured object. After sectioning the 3D object into N consecutive slices, all N slices are scanned using an optical digital scanner, enabling for performing the MS measurements. Scanning the individual sections results into low-resolution images, which define the base coordinate system for the whole pipeline. The scanned images conclude the information from the spatial (MRI) and the mass spectrometric (MALDI-MSI) dimension and are used for the spatial three-dimensional reconstruction of the object performed by image registration techniques. Different strategies for automatic serial image registration applied to MS datasets are outlined in detail. The third image modality is histology driven, i.e. a digital scan of the histological stained slices in high-resolution. After fusion of reconstructed scan images and MRI the slice-related coordinates of the mass spectra can be propagated into 3D-space. After image registration of scan images and histological stained images, the anatomical information from histology is fused with the mass spectra from MALDI-MSI. As a result of the described pipeline we have a set of 3 dimensional images representing the same anatomies, i.e. the reconstructed slice scans, the spectral images as well as corresponding clustering results, and the acquired MRI. Great emphasis is put on the fact that the co-registered MRI providing anatomical details improves the interpretation of 3D MALDI images. The ability to relate mass spectrometry derived molecular information with in vivo and in vitro imaging has potentially important implications. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

Fast proton spectroscopic imaging with high signal-to-noise ratio: spectroscopic RARE.

A new fast spectroscopic imaging (SI) method is presented which is based on spatial localization by the fast MRI method of rapid acquisition with relaxation enhancement (RARE) and encoding of the chemical shift information by shifting the position of a refocusing 180° pulse in a series of measurements. This method is termed spectroscopic RARE. In contrast to spectroscopic ultrafast low‐angle RARE (U‐FLARE), the formation of two echo families (odd and even) is suppressed by using a train of 180° RF pulses with an internal four‐step phase cycle. By this means a high signal‐to‐noise ratio (SNR) per unit measurement time is obtained, because the separation of odd and even echoes, as well as dummy echoes to stabilize the echo amplitudes, is not needed anymore. The method is of particular interest for detecting signals of coupled spins, as effective homonuclear decoupling can be achieved by use of constant evolution time chemical shift encoding. The pulse sequence was implemented on a 4.7 T imaging system, tested on phantoms, and applied to the healthy rat brain in vivo. Spectroscopic RARE is particularly useful if T  *2 ≪ T2, which is typically fulfilled for in vivo proton SI measurements at high magnetic field strength. Magn Reson Med 47:523–528, 2002.