Cristina Ramona Cudalbu

Ammonia toxicity to the brain

Hyperammonemia can be caused by various acquired or inherited disorders such as urea cycle defects. The brain is much more susceptible to the deleterious effects of ammonium in childhood than in adulthood. Hyperammonemia provokes irreversible damage to the developing central nervous system: cortical atrophy, ventricular enlargement and demyelination lead to cognitive impairment, seizures and cerebral palsy. The mechanisms leading to these severe brain lesions are still not well understood, but recent studies show that ammonium exposure alters several amino acid pathways and neurotransmitter systems, cerebral energy metabolism, nitric oxide synthesis, oxidative stress and signal transduction pathways. All in all, at the cellular level, these are associated with alterations in neuronal differentiation and patterns of cell death. Recent advances in imaging techniques are increasing our understanding of these processes through detailed in vivo longitudinal analysis of neurobiochemical changes associated with hyperammonemia. Further, several potential neuroprotective strategies have been put forward recently, including the use of NMDA receptor antagonists, nitric oxide inhibitors, creatine, acetyl-L-carnitine, CNTF or inhibitors of MAPKs and glutamine synthetase. Magnetic resonance imaging and spectroscopy will ultimately be a powerful tool to measure the effects of these neuroprotective approaches.

1H NMR spectroscopy of rat brain in vivo at 14.1Tesla: improvements in quantification of the neurochemical profile.

Ultra-short echo-time proton single voxel spectra of rat brain were obtained on a 14.1T 26 cm horizontal bore system. At this field, the fitted linewidth in the brain tissue of adult rats was about 11 Hz. New, separated resonances ascribed to phosphocholine, glycerophosphocholine and N-acetylaspartate were detected for the first time in vivo in the spectral range of 4.2-4.4 ppm. Moreover, improved separation of the resonances of lactate, alanine, gamma-aminobutyrate, glutamate and glutathione was observed. Metabolite concentrations were estimated by fitting in vivo spectra to a linear combination of simulated spectra of individual metabolites and a measured spectrum of macromolecules (LCModel). The calculated concentrations of metabolites were generally in excellent agreement with those obtained at 9.4T. These initial results further indicated that increasing magnetic field strength to 14.1T enhanced spectral resolution in (1)H NMR spectroscopy. This implies that the quantification of the neurochemical profile in rodent brain can be achieved with improved accuracy and precision.

Marker-independent identification of glioma-initiating cells.

Tumor-initiating cells with stem cell properties are believed to sustain the growth of gliomas, but proposed markers such as CD133 cannot be used to identify these cells with sufficient specificity. We report an alternative isolation method purely based on phenotypic qualities of glioma-initiating cells (GICs), avoiding the use of molecular markers. We exploited intrinsic autofluorescence properties and a distinctive morphology to isolate a subpopulation of cells (FL1+) from human glioma or glioma cultures. FL1+ cells are capable of self-renewal in vitro, tumorigenesis in vivo and preferentially express stem cell genes. The FL1+ phenotype did not correlate with the expression of proposed GIC markers. Our data propose an alternative approach to investigate tumor-initiating potential in gliomas and to advance the development of new therapies and diagnostics.

Feasibility of in vivo N-15 MRS detection of hyperpolarized N-15 labeled choline in rats

The increase of total choline in tumors has become an important biomarker in cancer diagnosis. Choline and choline metabolites can be measured in vivo and in vitro using multinuclear MRS. Recent in vivo(13)C MRS studies using labeled substrates enhanced via dynamic nuclear polarization demonstrated the tremendous potential of hyperpolarization for real-time metabolic studies. The present study demonstrates the feasibility of detecting hyperpolarized (15)N labeled choline in vivo in a rat head at 9.4 T. We furthermore report the in vitro (172 +/- 16 s) and in vivo (126 +/- 15 s) longitudinal relaxation times. We conclude that with appropriate infusion protocols it is feasible to detect hyperpolarized (15)N labeled choline in live animals.

Hyperpolarized lithium-6 as a sensor of nanomolar contrast agents

Lithium is widely used in psychotherapy. The 6Li isotope has a long intrinsic longitudinal relaxation time T1 on the order of minutes, making it an ideal candidate for hyperpolarization experiments. In the present study we demonstrated that lithium‐6 can be readily hyperpolarized within 30 min, while retaining a long polarization decay time on the order of a minute. We used the intrinsically long relaxation time for the detection of 500 nM contrast agent in vitro. Hyperpolarized lithium‐6 was administered to the rat and its signal retained a decay time on the order of 70 sec in vivo. Localization experiments imply that the lithium signal originated from within the brain and that it was detectable up to 5 min after administration. We conclude that the detection of submicromolar contrast agents using hyperpolarized NMR nuclei such as 6Li may provide a novel avenue for molecular imaging. Magn Reson Med, 2009.

Comparison of T1 relaxation times of the neurochemical profile in rat brain at 9.4 tesla and 14.1 tesla.

Knowledge of T1 relaxation times can be important for accurate relative and absolute quantification of brain metabolites, for sensitivity optimizations, for characterizing molecular dynamics, and for studying changes induced by various pathological conditions. 1H T1 relaxation times of a series of brain metabolites, including J‐coupled ones, were determined using a progressive saturation (PS) technique that was validated with an adiabatic inversion‐recovery (IR) method. The 1H T1 relaxation times of 16 functional groups of the neurochemical profile were measured at 14.1T and 9.4T. Overall, the T1 relaxation times found at 14.1T were, within the experimental error, identical to those at 9.4T. The T1s of some coupled spin resonances of the neurochemical profile were measured for the first time (e.g., those of γ‐aminobutyrate [GABA], aspartate [Asp], alanine [Ala], phosphoethanolamine [PE], glutathione [GSH], N‐acetylaspartylglutamate [NAAG], and glutamine [Gln]). Our results suggest that T1 does not increase substantially beyond 9.4T. Furthermore, the similarity of T1 among the metabolites (∼1.5 s) suggests that T1 relaxation time corrections for metabolite quantification are likely to be similar when using rapid pulsing conditions. We therefore conclude that the putative T1 increase of metabolites has a minimal impact on sensitivity when increasing B0 beyond 9.4T. Magn Reson Med, 2009.

Handling Macromolecule Signals in the Quantification of the Neurochemical Profile

In vivo localized proton magnetic resonance spectroscopy (1H MRS) became a powerful and unique technique to non-invasively investigate brain metabolism of rodents and humans. The main goal of 1H MRS is the reliable quantification of concentrations of metabolites (neurochemical profile) in a well-defined region of the brain. The availability of very high magnetic field strengths combined with the possibility of acquiring spectra at very short echo time have dramatically increased the number of constituents of the neurochemical profile. The quantification of spectra measured at short echo times is complicated by the presence of macromolecule signals of particular importance at high magnetic fields. An error in the macromolecule estimation can lead to substantial errors in the obtained neurochemical profile. The purpose of the present review is to overview methods of high field 1H MRS with a focus on the metabolite quantification, in particular in handling signals of macromolecules. Three main approaches of handling signals of macromolecules are described, namely mathematical estimation of macromolecules, measurement of macromolecules in vivo, and direct acquisition of the in vivo spectrum without the contribution of macromolecules.

Cerebral glutamine metabolism under hyperammonemia determined in vivo by localized 1H and 15N NMR spectroscopy

Brain glutamine synthetase (GS) is an integral part of the glutamate—glutamine cycle and occurs in the glial compartment. In vivo Magnetic Resonance Spectroscopy (MRS) allows noninvasive measurements of the concentrations and synthesis rates of metabolites. 15N MRS is an alternative approach to 13C MRS. Incorporation of labeled 15N from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate—glutamine cycle. To measure 15N incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel 15N pulse sequence to simultaneously detect, for the first time, [5-15N]Gln and [2-15N]Gln + Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized 1H spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of 1H and 15N MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30 ± 0.050 μmol/g per minute), apparent neurotransmission (0.26 ± 0.030 μmol/g per minute), glutamate dehydrogenase (0.029 ± 0.002 μmol/g per minute), and net glutamine accumulation (0.033 ± 0.001 μmol/g per minute). These results showed an increase of GS and net glutamine accumulation under hyperammonemia, supporting the concept of their implication in cerebral ammonia detoxification.

Proton and Phosphorus Magnetic Resonance Spectroscopy of a Mouse Model of Alzheimer's Disease

The development of new diagnostic criteria for Alzheimers disease (AD) requires new in vivo markers reflecting early pathological changes in the brain of patients. Magnetic resonance (MR) spectroscopy has been shown to provide useful information about the biochemical changes occurring in AD brain in vivo. The development of numerous transgenic mouse models of AD has facilitated the evaluation of early biomarkers, allowing researchers to perform longitudinal studies starting before the onset of the pathology. In addition, the recent development of high-field animal scanners enables the measurement of brain metabolites that cannot be reliably quantified at lower magnetic fields. In this report, we studied a new transgenic mouse model of AD, the 5xFAD model, by in vivo proton and phosphorus MR spectroscopy. This model, which is characterized by an early-onset and a robust amyloid pathology, developed changes in the neurochemical profile, which are typical in the human disease, i.e., an increase in myo-inositol and a decrease in N-acetylaspartate concentrations, as early as in the 40th week of age. In addition, a significant decrease in the γ-aminobutyrate concentration was observed in transgenic mice at this age compared to controls. The pseudo-first-order rate constant of the creatine kinase reaction as well as relative concentrations of phosphorus-containing metabolites were not changed significantly in the 36 and 72-week old transgenic mice. Overall, these results suggest that mitochondrial activity in the 5 × FAD mice is not substantially affected but that the model is relevant for studying early biomarkers of AD.

Quantification of the neurochemical profile using simulated macromolecule resonances at 3 T

The broad resonances underlying the entire 1H NMR spectrum of the brain, ascribed to macromolecules, can influence metabolite quantification. At the intermediate field strength of 3 T, distinct approaches for the determination of the macromolecule signal, previously used at either 1.5 or 7 T and higher, may become equivalent. The aim of this study was to evaluate, at 3 T for healthy subjects using LCModel, the impact on the metabolite quantification of two different macromolecule approaches: (i) experimentally measured macromolecules; and (ii) mathematically estimated macromolecules. Although small, but significant, differences in metabolite quantification (up to 23% for glutamate) were noted for some metabolites, 10 metabolites were quantified reproducibly with both approaches with a Cramer–Rao lower bound below 20%, and the neurochemical profiles were therefore similar. We conclude that the mathematical approximation can provide sufficiently accurate and reproducible estimation of the macromolecule contribution to the 1H spectrum at 3 T. Copyright