Biomedical subjects

D Leibfritz

Publications and source records attributed to D Leibfritz.

At least 19 recordsLinked to original sources

PubMed1997

Study of the interactions of cadmium and zinc ions with cellular calcium homoeostasis using 19F-NMR spectroscopy.

The effects of the heavy-metal ions Cd2+ and Zn2+ on the homoeostasis of intracellular free Ca2+ in E367 neuroblastoma cells were examined using 19F-NMR spectroscopy with the fluorinated chelator probe 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N', N'-tetra-acetic acid (5F-BAPTA). First, the technique was used to quantify the uptake and intracellular free concentrations of the heavy metals after treatment of the cells with 20 microM CdCl2 or 100 microM ZnCl2. Secondly, metal-induced transients in intracellular free Ca2+ were recorded. Addition of 20 microM CdCl2, but not 100 microM ZnCl2, evoked a transient increase in Ca2+ from a resting level of 84 nM to approx. 190 nM within 15 min after addition of the metal. Zn2+ at 20 microM completely prevented the induction of a Ca2+ transient by Cd2+. Ca2+ was mobilized by Cd2+ from intracellular organelles, since depletion of these stores by thapsigargin abolished the effect of the toxic metal. Furthermore, 20 microM Cd2+ evoked a transient rise in cellular Ins(1,4,5)P3, reaching a maximum level within 5 min after addition of the metal. These results demonstrate that perturbation of the Ins(1,4,5)P3/Ca2+ messenger system is an early and discrete cellular effect of Cd2+.

Animals↗

PubMed1997

Interactions of triethyltin-chloride (TET) with the energy metabolism of cultured rat brain astrocytes: studies by multinuclear magnetic resonance spectroscopy.

The effect of triethyltin-chloride (TET), a highly neurotoxic compound, on the cellular metabolism of rat brain astrocytes in vitro was examined by nuclear magnetic resonance (NMR) spectroscopy. 5-week-old cultures were exposed to TET (0.2-40 microM) either for (1) acute (3h), (2) 24 h, or (3) chronic treatment (8 d). Cells were labeled with 1-(13)C-glucose, cell extracts were prepared and 31P, 1H, and 13C spectra were analyzed. Cytotoxic effects of TET were assessed by vital dye uptake assay using neutral red (NR) and by exclusion of trypan blue (TB). Cells were examined ultrastructurally by electron microscopy. The data show that the major target of TET at concentrations already causing morphological effects on cultured astrocytes is not the energy metabolism, but that TET rather alters the intracellular concentrations of organic osmolytes, such as myo-inositol, taurine and hypotaurine, which are part of the control of ion and volume regulation and osmotic balance in astrocytes.

Animals↗

PubMed1996

Evaluation of the effects of immunosuppressants on neuronal and glial cells in vitro by multinuclear magnetic resonance spectroscopy.

The use of the undecapeptide cyclosporine and the macrolide tacrolimus as immunosuppressants in transplantation medicine and for the therapy of immune diseases often provokes side effects, among the most important one is neurotoxicity. Changes in the cellular metabolism of glial cells (C6 rat glioma), neuronal cells (N1E-115 mouse neuroblastoma) and primary glia cells (isolated from rats) after addition of cyclosporine and tacrolimus were investigated using 1H-, 13C- and 31P-NMR spectroscopy in vitro. Cells were exposed to various concentrations of the drugs from 3 h to 42 days. The immunosuppressants (cyclosporine IC50 : 55 mumol/l; tacrolimus IC50 : 47 mumol/l) inhibited cell proliferation in a concentration- and time-dependent fashion. Multinuclear NMR studies of PCA extracts of drug-treated cells showed a significant deterioration in the energy status (a decreasing level of PCr : -46 +/- 11%; an increasing NDP/NTP ratio: +136 +/- 4% and an increasing level of Pi : +248 +/- 15%; mean +/- standard deviation). It also showed decreasing concentrations of major cell metabolites like NAA (-59 +/- 12%) in neuroblastoma cells and myo-inositol (-47 +/- 6%) in glia cells compared with untreated controls. Immunosuppressive treatment caused a large reduction of taurine (-36 +/- 12%) and glutamate (-68 +/- 10%) in all cell cultures, whereas intermediates of phospholipid biosynthesis (PE: +59 +/- 13%; PC: +127 +/- 27%;) and breakdown (GPE: +215 +/- 24%; GPC: +245 +/- 17%) increased. No significant differences were observed between the two immunosuppressants. The toxic effects of immunosuppressants on cell cultures are in line with MRI studies of brain oedema observed in patients under immunosuppressive treatment.

Animals↗

PubMed1996

Status of the neonatal rat brain after NMDA-induced excitotoxic injury as measured by MRI, MRS and metabolic imaging.

Intrastriatal injection of the excitotoxin N-methyl-D-aspartate (NMDA) in neonatal rat brain resulted in an acute ipsilateral decrease of the apparent diffusion coefficient (ADC) of brain tissue water, as measured with diffusion-weighted MRI. The early diffusion changes were accompanied by only mild changes in the overall metabolic status as measured by in vivo 1H MRS and 31P MRS and metabolic imaging of brain sections. Minimal decreases in the high-energy phosphate levels and a small hemispheric acidosis were observed in the first 6 h after NMDA administration. In addition, there was very modest lactate accumulation. Twenty-four hours after the induction of the excitotoxic injury the tissue energy status was still only moderately affected, whereas an overall decrease of 1H MRS-detected brain metabolites was found. Treatment with the non-competitive NMDA-antagonist MK-801 given within 90 min after NMDA injection rapidly reversed the NMDA-induced changes in the entire ipsilateral hemisphere. The effect of the competitive NMDA-antagonist D-CPPene was restricted to the cortical areas and was accomplished on a slower time scale. Our results indicate that; (i) early excitotoxicity in the neonatal rat brain does not lead to profound changes in the metabolic status; and (ii) brain tissue water ADC changes are not necessarily associated with a metabolic energy failure.

Animals↗

PubMed1996

Fast proton spectroscopic imaging employing k-space weighting achieved by variable repetition times.

A k-space weighted spectroscopic imaging (SI) method is presented that allows a reduction in the total data acquisition time by up to 55% compared with standard SI. The k-space weighting is achieved by varying the repetition time, thus realizing an inherent apodization that corresponds to a circularly symmetric generalized Hamming filter. The flip angle is varied with the repetition time to enhance the signal-to-noise ratio. These techniques were employed using a short echo time of 10 ms. In vivo measurements on healthy rat brain at 4.7 T were conducted, obtaining two-dimensional spectroscopic imaging data from a 25 x 25 circularly reduced k-space area in as little as 5 min. The signal-to-noise ratio is sufficiently high to detect J-coupled resonances such as myo-inositol or glutamate/glutamine, demonstrating the ability to combine short acquisition times with comprehensive metabolic information. The T1 dependency of the apodization and the corresponding point spread function was evaluated by computer simulations. The achievable signal-to-noise ratio per unit time was compared with standard SI giving a parameter-dependent advantage of approximately 20% of the standard SI method.

Animals↗

PubMed1996

Adaptation of cellular metabolism to anisosmotic conditions in a glial cell line, as assessed by 13C-NMR spectroscopy.

13C-NMR spectroscopy of perchloric acid and lipid extracts of F98 glioma cells showed that volume-regulatory processes under anisosmotic conditions were accompanied by marked alterations in cellular metabolism. Production of alanine, glutamate, and glycine from [U-13C]-glucose is decreased under hypotonic stress and is oppositely increased under hypertonic stress. In contrast, degradation of these molecules is raised under hypotonic conditions and reduced under hypertonic conditions. Furthermore, phospholipid synthesis is decreased under hypertonic stress and increased under hypotonic stress. Obviously, glial metabolism is directed under hypertonic conditions to maintain a high level of small, osmotically active molecules, whereas under hypotonic conditions molecular fragments are increasingly incorporated into the phospholipids and so do not contribute to the osmotic pressure. The latter is evoked by the activation of membrane synthesis process to compensate for stretching and/or damaging of the membranes due to cell swelling.

Adaptation, Physiological↗

PubMed1996

Immobilization of primary astrocytes and neurons for online monitoring of biochemical processes by NMR.

Immobilization of primary astrocytes and cerebellar granule neurons in basement membrane gel threads and on porous microcarriers, respectively, is described. Five different porous microcarriers were examined for their capabilities to support maturation of neurons, as evaluated by determinations of protein content and glucose uptake. These systems proved suitable for perfusion in the NMR tube for long periods of time. The energetic status of the cells was monitored by 31P-NMR, and a very good temporal resolution (less than 30 min) was achieved with superfused astrocytes; thus, this provides a valuable method for real time NMR investigation of biochemical processes in primary astrocytes. Significantly longer acquisition times (approximately 8 h) were required in order to obtain a spectrum of neurons with a reasonably good signal to noise ratio, even so, the results presented here and obtained with this difficult system are very encouraging.

Animals↗

PubMed1995

On the use of two-dimensional-J NMR measurements for in vivo proton MRS: measurement of homonuclear decoupled spectra without the need for short echo times.

The potential of two-dimensional (2D)-J NMR for in vivo proton MRS is examined. Single voxel measurements on the rat brain were performed at 4.7 T using point-resolved spectrocopy localization with a voxel size of 64 microliter and total measuring times of 10-15 min. It is shown that a series of measurements with only 16 or fewer different echo times (TE) enables good signal localization in the f1 axis corresponding to the coupling patterns. For data evaluation, the 2D-J NMR spectrum as well as cross-sections at given f1 values and projections onto the f2 axis are used. A comparison between cross-section spectra taken at different f1 values may help to solve problems of peak assignment. The projection of the 2D magnitude spectrum onto the f2 axis corresponds to a homonuclear decoupled 1D proton spectrum. Because the T2 relaxation times of several coupled resonances (e.g., myo-inositol and glutamate) are rather long, only minor losses in the quality of the projection spectra occur if the measurements with short TE (< or = 50 ms) are not used for data processing. Thus, homonuclear decoupled proton spectra detecting uncoupled and several coupled resonances can be measured with high quality in vivo, even on MR systems that are not equipped with actively shielded gradients, prohibiting data acquisition with TEs of 50 ms or less.

Animals↗

PubMed1995

A fast method for in vivo lactate imaging.

A robust method for fast lactate imaging is presented using a combination of a lactate editing sequence and a one-shot imaging experiment. The lactate editing method is based on manipulation of the phase of the lactate CH3 signal via J-modulation. This is applied as a preparation experiment to the U-FLARE imaging sequence. Phantom experiments are presented in which the quality of water and lipid suppression is established. Edited lactate images with a spatial resolution of 3 microL and total measuring time of 15.8 min are shown. These were obtained from a hemispherical ischemia in the gerbil brain. The images are compared with diffusion-weighted water images.

Animals↗

PubMed1995

Changes in organic solutes, volume, energy state, and metabolism associated with osmotic stress in a glial cell line: a multinuclear NMR study.

Diffusion-weighted in vivo 1H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM. 1H-, 13C- and 31P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo 31P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.

Animals↗

PubMed1995

Quality assessment in in vivo NMR spectroscopy: I. Introduction, objectives, and activities.

By enabling noninvasive measurements of tissue biochemistry, nuclear magnetic resonance spectroscopy (MRS) provides a unique means of characterizing tissues. Differences in equipment, techniques, and methodology between different laboratories cause major difficulties when comparing results, whether from measurements of tissue metabolism, or from the effects of different therapies. This is of concern in critically evaluating work from different laboratories and centres, causing potential difficulties in reproducing results, limiting the establishment of MRS as a standard method of diagnosis and of characterising disease and response to therapy in the laboratory and clinic. It also poses particular problems in establishing the multicentre clinical trials of MRS that are now required to provide adequate statistical power in confirming the encouraging preliminary clinical observations. These difficulties arise principally from imperfect localization of signal from selected regions of interest in the body, and from the subsequent analyses of the MRS spectra. Improvement is possible by establishing agreed procedures for test measurements and for data analysis, and by using appropriate test objects and test substances to establish the quality of measurements. A concerted research project on characterisation of biological tissues by NMR, principally concerned with MR imaging (MRI), was activated in 1984 by the European Economic Community as part of its third Medical and Health Research Programme, under the auspices of the Biomedical Engineering Concerted Actions' Committee (COMAC-BME). In 1988, this project was prolonged for 5 years, when the programme was expanded to encompass MRS.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗

PubMed1995

Parametric multiecho proton spectroscopic imaging: application to the rat brain in vivo.

A parametric multiecho variant of proton spectroscopic imaging (SI) is presented using a multiecho SI sequence with uniform phase-encoding of all echoes within each echo train. The acquisition of SI data sets at different echo times (TE) increases the amount of information obtained within the same total measuring time as in standard SI measurements. The gain in information can be used: (a) to choose the most appropriate TE for each metabolite signal with respect to T2, spin coupling, or problems caused by peak overlap; (b) to measure the relaxation time T2 of metabolite signals with high spatial resolution; or (c) to improve the signal-to-noise ratio for metabolite signals with long T2 values by adding spectra calculated from consecutive echoes. The method was tested in vivo on healthy rat brain and applied to study metabolic changes in rat brain lesions.

Animals↗

PubMed1995

Evolution of regional changes in apparent diffusion coefficient during focal ischemia of rat brain: the relationship of quantitative diffusion NMR imaging to reduction in cerebral blood flow and metabolic disturbances.

Middle cerebral artery occlusion was performed in rats while the animals were inside the nuclear magnetic resonance (NMR) tomograph. Successful occlusion was confirmed by the collapse of amplitude on an electrocorticogram. The ultrafast NMR imaging technique UFLARE was used to measure the apparent diffusion coefficient (ADC) immediately after the induction of cerebral ischemia. ADC values of normal cortex and caudate-putamen were 726 +/- 22 x 10(-6) mm2/s and 659 +/- 17 x 10(-6) mm2/s, respectively. Within minutes of occlusion, a large territory with reduced ADC became visible in the ipsilateral hemisphere. Over the 2 h observation period, this area grew continuously. Quantitative analysis of the ADC reduction in this region showed a gradual ADC decrease from the periphery to the core, the lowest ADC value amounting to about 60% of control. Two hours after the onset of occlusion, the regional distribution of ATP and tissue pH were determined with bioluminescence and fluorescence techniques, respectively. There was a depletion of ATP in the core of the ischemic territory (32 +/- 20% of the hemisphere) and an area of tissue acidosis (57 +/- 19% of the hemisphere) spreading beyond that of ATP depletion. Regional CBF (rCBF) was measured autoradiographically with the iodo[14C]antipyrine method. CBF gradually decreased from the periphery to the ischemic core, where it declined to values as low as 5 ml 100 g-1. When reductions in CBF and in ADC were matched to the corresponding areas of energy breakdown and of tissue acidosis, the region of energy depletion corresponded to a threshold in rCBF of 18 +/- 14 ml 100 g-1 min-1 and to an ADC reduction to 77 +/- 3% of control. Tissue acidosis corresponded to a flow value below 31 +/- 11 ml 100 g-1 min-1 and to an ADC value below 90 +/- 4% of control. Thus, the quantification of ADC in the ischemic territory allows the distinction between a core region with total breakdown of energy metabolism and a corona with normal energy balance but severe tissue acidosis.

Adenosine Triphosphate↗

PubMed1994

Magnetization transfer affects the proton creatine/phosphocreatine signal intensity: in vivo demonstration in the rat brain.

The effect of off-resonance preirradiation on proton spectra acquired from the healthy rat brain at 4.7 T is examined using a PRESS sequence. The creatine/phosphocreatine (Cr/PCr) signal at 3.0 ppm decreases in signal intensity for offset frequencies between +/- 10 kHz. This cannot be explained by RF bleedover, but is attributed to magnetization transfer between two pools of Cr/PCr, one with a long T2 relaxation time giving the observed NMR signal, the other corresponding to a broad resonance line being completely or partially saturated by off-resonance preirradiation. Possible interpretations of these results are discussed.

Animals↗

PubMed1994

Double-echo multislice proton spectroscopic imaging using Hadamard slice encoding.

Simultaneous multislice proton spectroscopic imaging (SI) is presented using a pulse sequence with multifrequency selective RF excitation and Hadamard encoding in the slice direction, and conventional Fourier phase encoding in the in-plane directions. Double-echo data acquisition is used to increase the spectral information of the experiment. Tests on a phantom demonstrate the quality of the slice selection. Results of in vivo measurements of the healthy rat brain show that spectra with a high signal-to-noise ratio can be acquired from four slices within 32 min. The measurements were performed at 4.7 T using a field of view of 32 x 32 mm2, a slice thickness of 3 mm, and a voxel size of 12 microliters. The proposed method is a useful alternative to sequential multislice SI and 3D SI. Furthermore, it is possible to combine sequential and simultaneous multislice SI.

Animals↗

PubMed1994

Detection of apparent restricted diffusion in healthy rat brain at short diffusion times.

The application of bipolar diffusion sensitizing gradient pulses to significantly reduce the diffusion time is described. This approach is combined with the rapid U-FLARE imaging sequence. Three diffusion-sensitized types of experiments are compared and their suitability for detecting restricted diffusion is discussed. Experiments using a modification of the diffusion weighting by varying the diffusion time between 1.6 and 6.0 ms obtained nonmonoexponential signal attenuation curves from both healthy brains and postmortem. This behavior is indicative of restricted diffusion, but as it is detectable only at short diffusion times, in contrast to a restriction due to impermeable barriers, we have termed this "apparent restriction."

Animals↗

PubMed1994

Regulation of intracellular pH in neuronal and glial tumour cells, studied by multinuclear NMR spectroscopy.

The effect of extracellular pH (pHe) on intracellular pH (pHi) and cellular metabolism was examined by multinuclear NMR spectroscopy of cells in vivo and in vitro. A decrease in pHe from 7.4 to 6.4 led to a significant drop in pHi, in both neuronal and glial tumour cells, as detected by in vivo 31P NMR of cells embedded in basement membrane gel threads. A more than 50% decrease in both the phosphocreatine (PCr) level and derivatives of glycolysis (i.e., glycerol 3-phosphate) was observed, concomitantly to the fall in pHi. A 50% decrease in intracellular lactate levels was seen in in vivo 1H NMR spectra under these conditions. Reperfusion with fresh medium (pHe 7.4) resulted in the full recovery of pHi, simultaneously with an increase in both PCr and intracellular lactate back to their control levels. Perchloric acid and lipid extract measurements confirmed the observations made by in vivo 31P and 1H NMR spectroscopy and further showed a decrease both in tricarboxylic acid cycle activity and phospholipid synthesis. The data revealed no significant differences between the neuronal and glial tumour cells investigated. pHi measurements in the presence of inhibitors of the various pH regulatory mechanisms showed that the Na+/H+ exchanger, the carbonic anhydrase and at least one of the bicarbonate-transport systems are involved in pH regulation of both cell types. The results suggest that Na+/H+ exchange is the preferred mechanism by which both neuronal and glial cells regulate their pHi after extracellular acidification.

Animals↗

PubMed1994

Health and infarcted brain tissues studied at short diffusion times: the origins of apparent restriction and the reduction in apparent diffusion coefficient.

The significance of NMR water diffusion measurements performed at short diffusion times (< 10 ms) for brain tissue is examined. An apparent restriction to diffusion for both healthy and cytotoxically edematous tissue is shown: cytotoxic edema lengthens the diffusion time at which this phenomenon is visible. The dramatic reduction in apparent diffusion coefficient (ADC) observed in the core of cytotoxic edema is explained in terms of the enclosure of extracellular water in non-contiguous pockets in conjunction with the shift of water from the extra-to the intracellular space. The model presented provides an explanation for the ADC reduction without recourse to changes in the cell membrane permeability to water, or unrealistic values for the extra- and intracellular diffusion coefficients.

Animals↗