Lower Lactate Levels and Lower Intracellular pH in Patients with IDH-Mutant versus Wild-Type Gliomas.

BACKGROUND AND PURPOSE: Preclinical evidence points toward a metabolic reprogramming in isocitrate dehydrogenase (IDH) mutated tumor cells with down-regulation of the expression of genes that encode for glycolytic metabolism. We noninvasively investigated lactate and Cr concentrations, as well as intracellular pH using 1H/phosphorus 31 (31P) MR spectroscopy in a cohort of patients with gliomas. MATERIALS AND METHODS: Thirty prospectively enrolled, mostly untreated patients with gliomas met the spectral quality criteria (World Health Organization II [n = 7], III [n = 16], IV [n = 7]; IDH-mutant [n = 23]; IDH wild-type [n = 7]; 1p/19q codeletion [n = 9]). MR imaging protocol included 3D 31P chemical shift imaging and 1H single-voxel spectroscopy (point-resolved spectroscopy sequence at TE = 30 ms and TE = 97 ms with optimized echo spacing for detection of 2-hydroxyglutarate) from the tumor area. Values for absolute metabolite concentrations were calculated (phantom replacement method). Intracellular pH was determined from 31P chemical shift imaging. RESULTS: At TE = 97 ms, lactate peaks can be fitted with little impact of lipid/macromolecule contamination. We found a significant difference in lactate concentrations, lactate/Cr ratios, and intracellular pH when comparing tumor voxels of patients with IDH-mutant with those of patients with IDH wild-type gliomas, with reduced lactate levels and near-normal intracellular pH in patients with IDH-mutant gliomas. We additionally found evidence for codependent effects of 1p/19q codeletion and IDH mutations with regard to lactate concentrations for World Health Organization tumor grades II and III, with lower lactate levels in patients exhibiting the codeletion. There was no statistical significance when comparing lactate concentrations between IDH-mutant World Health Organization II and III gliomas. CONCLUSIONS: We found indirect evidence for metabolic reprogramming in IDH-mutant tumors with significantly lower lactate concentrations compared with IDH wild-type tumors and a near-normal intracellular pH.

Sex-associated differences in mitochondrial function in human peripheral blood mononuclear cells (PBMCs) and brain.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia, and it affects more women than men. Mitochondrial dysfunction (MD) plays a key role in AD, and it is detectable at an early stage of the degenerative process in peripheral tissues, such as peripheral mononuclear blood cells (PBMCs). However, whether these changes are also reflected in cerebral energy metabolism and whether sex-specific differences in mitochondrial function occur are not clear. Therefore, we estimated the correlation between mitochondrial function in PBMCs and brain energy metabolites and examined sex-specific differences in healthy participants to elucidate these issues. METHODS: The current pilot study included 9 male and 15 female healthy adults (mean age 30.8 ± 7.1 years). Respiration and activity of mitochondrial respiratory complexes were measured using a Clarke-electrode (Oxygraph-2k system), and adenosine triphosphate (ATP) levels were determined using a bioluminescence-based assay in isolated PBMCs. Citrate synthase activity as a mitochondrial marker was measured using a photometric assay. Concentrations of brain energy metabolites were quantified in the same individuals using 1H-magnetic resonance spectroscopy (MRS). RESULTS: We detected sex-associated differences in mitochondrial function. Mitochondrial complexes I, I+II, and IV and uncoupled respiration and electron transport system (ETS) capacity in PBMCs isolated from blood samples of females were significantly (p < 0.05; p < 0.01) higher compared to males. ATP levels in the PBMCs of female participants were approximately 10% higher compared to males. Citrate synthase (CS) activity, a marker of mitochondrial content, was significantly (p < 0.05) higher in females compared to males. Sex-associated differences were also found for brain metabolites. The N-acetylaspartate (NAA) concentration was significantly higher in female participants compared to males in targeted regions. This difference was observed in white matter (WM) and an area with a high percentage (> 50%) of gray matter (GM) (p < 0.05; p < 0.01). The effect sizes indicated a strong influence of sex on these parameters. Sex-associated differences were found in PBMCs and brain, but the determined parameters were not significantly correlated. CONCLUSIONS: Our study revealed sex-associated differences in mitochondrial function in healthy participants. The underlying mechanisms must be elucidated in more detail, but our study suggests that mitochondrial function in PBMCs is a feasible surrogate marker to detect differences in mitochondrial function and energy metabolism in humans and it underscores the necessity of sex-specific approaches in therapies that target mitochondrial dysfunction.

Effects of aerobic exercise on brain metabolism and grey matter volume in older adults: results of the randomised controlled SMART trial.

There is mounting evidence that aerobic exercise has a positive effect on cognitive functions in older adults. To date, little is known about the neurometabolic and molecular mechanisms underlying this positive effect. The present study used magnetic resonance spectroscopy and quantitative MRI to systematically explore the effects of physical activity on human brain metabolism and grey matter (GM) volume in healthy aging. This is a randomised controlled assessor-blinded two-armed trial (n=53) to explore exercise-induced neuroprotective and metabolic effects on the brain in cognitively healthy older adults. Participants (age >65) were allocated to a 12-week individualised aerobic exercise programme intervention (n=29) or a 12-week waiting control group (n=24). The main outcomes were the change in cerebral metabolism and its association to brain-derived neurotrophic factor (BDNF) levels as well as changes in GM volume. We found that cerebral choline concentrations remained stable after 12 weeks of aerobic exercise in the intervention group, whereas they increased in the waiting control group. No effect of training was seen on cerebral N-acetyl-aspartate concentrations, nor on markers of neuronal energy reserve or BDNF levels. Further, we observed no change in cortical GM volume in response to aerobic exercise. The finding of stable choline concentrations in the intervention group over the 3 month period might indicate a neuroprotective effect of aerobic exercise. Choline might constitute a valid marker for an effect of aerobic exercise on cerebral metabolism in healthy aging.

Brain energy metabolism in early MSA-P: A phosphorus and proton magnetic resonance spectroscopy study.

INTRODUCTION: Recently, mutations in the COQ2 gene, encoding for an enzyme involved in coenzyme Q10 biosynthesis, have been suggested to confer susceptibility risk for multiple system atrophy (MSA). Thus, the possible role of mitochondrial dysfunction in the pathophysiology of MSA has emerged. Here, we studied brain energy metabolism in vivo in early MSA-parkinsonism (MSA-P) patients and compared to healthy controls. METHODS: We have used combined phosphorus and proton magnetic resonance spectroscopy to measure high- and low-energy phosphates in the basal ganglia of early (Hoehn and Yahr stage I-III), probable MSA-P patients (N = 9) compared to healthy controls (N = 9). RESULTS: No significant changes in the high energy phosphates and other parameters reflecting the energy status of the cells were found in the basal ganglia of MSA-P patients compared to healthy controls. N-acetylaspartate was significantly reduced in MSA-P compared to healthy controls and correlated with the Unified Multiple System Atrophy Rating Scale. CONCLUSION: Brain energy metabolism in early MSA-P is not impaired, despite the presence of impaired neuronal integrity. This may imply that mitochondrial dysfunction may not play a primary role in the pathophysiology of MSA, at least in European populations.

¹H- and ¹³C-NMR spectroscopy of Thy-1-APPSL mice brain extracts indicates metabolic changes in Alzheimer's disease.

Biochemical alterations underlying the symptoms and pathomechanisms of Alzheimer's disease (AD) are not fully understood. However, alterations of glucose metabolism and mitochondrial dysfunction certainly play an important role. (1)H- and (13)C-NMR spectroscopy exhibits promising results in providing information about those alterations in vivo in patients and animals, especially regarding the mitochondrial tricarboxylic acid (TCA) cycle. Accordingly, transgenic mice expressing mutant human amyloid precursor protein (APP(SL))-serving as a model of neuropathological changes in AD-were examined with in vitro 1D (1)H- and 2D (1)H-(13)C-HSQC-NMR spectroscopy after oral administration of 1-(13)C-glucose and acquisition of brain material after 30 min. Perchloric acid extracts were measured using a 500 MHz spectrometer, providing more detailed information compared to in vivo spectra achievable nowadays. Area under curve (AUC) data of metabolite peaks were obtained and normalized in relation to the creatine signal, serving as internal reference. Besides confirming well-known metabolic alterations in AD like decreased N-acetylaspartate (NAA)/Creatine (Cr) ratio, new findings such as a decrease in phosphorylcholine (PC) are presented. Glutamate (Glu) and glutamine (Gln) concentrations were decreased while γ-aminobutyric acid (GABA) was elevated in Thy1-APP(SL) mice. (13)C-NMR spectroscopy revealed a shift in the Glx-2/Glx-4-ratio-where Glx represents a combined Glu/Gln-signal-towards Glx-2 in AD. These findings correlated well with the NAA/Cr-ratio. The Gln-4/Glu-4-ratio is altered in favor of Glu. Our findings suggest that glutamine synthetase (GS), which is predominantly present in glial cells may be impaired in the brain of Thy1-APP(SL) transgenic mice. Since GS is an ATP-dependent enzyme, mitochondrial dysfunction might contribute to reduced activity, which might also account for the increased metabolism of glutamate via the GABA shunt, a metabolic pathway to bypass intra-mitochondrial α-ketoglutarate-dehydrogenase, resulting in elevated GABA levels.

Metabolic changes in patients with aneurysmal subarachnoid hemorrhage apart from perfusion deficits: neuronal mitochondrial injury?

BACKGROUND AND PURPOSE: Neuronal damage in aSAH apart from perfusion deficits has been widely discussed. We aimed to test if cerebral injury occurs in aSAH independently from visible perfusion deficit by measuring cerebral metabolites in patients with aSAH without infarction or impaired perfusion. MATERIALS AND METHODS: We performed 3T MR imaging including (1)H-MR spectroscopy, DWI, and MR perfusion in 58 patients with aSAH and 11 age-matched and sex-matched control patients with incidental aneurysm. We compared changes of NAA, Cho, Glx, Lac, and Cr between all patients with aSAH and controls, between patients with and without visible perfusion deficit or infarction and controls, and between patients with and without visible perfusion deficit or infarction by using the Wilcoxon signed-rank test. RESULTS: We found that NAA significantly (P < .005) decreased in all patients with aSAH. Cho was significantly increased in all patients compared with controls (P < .05). In patients without impaired perfusion or infarction, Glx was significantly decreased compared with both controls (P = .005) and patients with impaired perfusion or infarction (P = .006). CONCLUSIONS: The significant decrease of NAA and Glx in patients with aSAH but without impaired perfusion or infarction strongly suggests global metabolic changes independent from visible perfusion deficits that might reflect neuronal mitochondrial injury. Further, impaired perfusion in aSAH seems to induce additional metabolic changes from increasing neuronal stress that might, to some extent, mask the global metabolic changes.

Objectification and quantification of the cognitive impairment from an existing HIV infection or HIV encephalopathy using magnetic resonance spectroscopy.

Some patients with HIV develop dementia. Using in vivo proton nuclear magnetic resonance ((1)H-NMR) spectroscopy, it is possible to measure the metabolic changes noninvasively. In this study, it is of interest to answer the clinically relevant question of whether magnetic resonance spectroscopy is suitable for the diagnosis of HIV encephalopathy. In total, 14 HIV-positive patients were investigated by means of localized (1)H-NMR spectroscopy in the following locations: (1) the mid-parietal gray matter, (2) the parietal white matter (PWM), and (3) the frontal white matter. All patients had no other brain diseases, apart from the HIV encephalopathy. The clinical extent of HIV encephalopathy of each patient was investigated using the following tests: (1) an electroencephalogram, (2) a neurological examination and psychiatric assessment, and (3) a psychometrical test. The spectroscopic changes in the PWM were more pronounced than those in the cortex, and the myo-inositol/creatine (mI/Cr) signal showed a clear increase in the cortex. Overall, the mI/Cr ratio emerged as the most reliable and earliest parameter to indicate an HIV encephalopathy.

Cutoff value of choline concentration reliably reveals high-grade brain tumors among other contrast-enhancing brain lesions.

BACKGROUND AND AIM: To evaluate whether there is a cutoff value for a metabolite concentration measured by 1 H MR spectroscopy (MRS), which can be used to differentiate malignant brain tumors (high-grade gliomas, primary CNS lymphomas [PCNSL] and metastases) from other contrast-enhancing lesions like low-grade gliomas and non-neoplastic lesions. MATERIAL AND METHODS: 1 H MRS was performed in 252 consecutive patients with space-occupying brain lesions which were enhanced with application of a contrast agent. Concentrations of N-acetyl-aspartate, total creatine, choline containing metabolites (total choline, tCho), lipids, and lactate were evaluated from the contrast-enhancing part of the lesions and from the normal appearing brain tissue. Linear discriminant analysis was used to find the best predictor for malignant brain tumors. In addition, receiver operating characteristic analysis (ROC) was performed to determine a cutoff value for the best predictor in detecting malignant brain tumors with a specificity of >95%. RESULTS: All brain tumors and 20 out of 47 nonneoplastic lesions were examined histopathologically. The remaining 27 diagnoses were based on MR imaging, clinical findings, and follow-up. The final diagnosis was 134 high-grade gliomas (WHO grade III/IV), 36 metastases, 9 PCNSL, 8 low-grade gliomas (WHO grade I/II), 34 infections, 9 infarctions, 2 hematomas, and 2 vasculitides. 18 patients were excluded due to insufficient spectral quality. The tCho concentration was the best predictor to differentiate malignant brain tumors from enhancing low-grade gliomas or non-neoplastic lesions (F=26.6 [df: 25.833], p<0.0005). The ROC revealed that a cutoff tCho value, based on an increase of ≥40% compared to normal, yielded a specificity of 100% and a sensitivity of 89.4% to correctly diagnose a malignant brain tumor. CONCLUSION: 1 H MRS reliably differentiates malignant brain tumors from other contrast-enhancing brain lesions. At least a 40% increase of tCho compared to normal brain tissue indicates a malignant tumor (WHO grade III/IV gliomas, PCNSL, metastases) with >90% specificity and sensitivity.

Neuroradiological viewpoint on the diagnostics of space-occupying brain lesions.

Conventional magnetic resonance (MR) imaging of space-occupying lesions may answer most of the questions concerning the diagnosis and subsequent treatment strategies if patient age, clinical and paraclinical findings are considered as well. However, crucial and relevant differential diagnoses require additional MR methods, such as diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI) and magnetic resonance spectroscopy (MRS). In necrotic ring-enhancing lesions DWI may detect inflammatory processes, whereas characteristics of the peritumoral area may help to distinguish between metastases and glioblastomas. In solid tumors DWI, PWI and MRS may also aid the differentiation between low-grade gliomas and malignant tumors, such as gliomas WHO (World Health Organization) grade III and IV and lymphomas. This review briefly explains special MR methods with respect to brain tumors and illustrates the diagnostic pathways necessary for supplying a reliable diagnosis as well as optimal pre-operative imaging of space-occupying brain lesions.

Combined 1H and 31P MR spectroscopic imaging: impaired energy metabolism in severe carotid stenosis and changes upon treatment.

OBJECT: To evaluate if combined (1)H and (31)P MR spectroscopic imaging (MRSI) before and after treatment of severe internal carotid artery (ICA) stenosis detects significant changes in energy metabolism in the basal ganglia of both hemispheres. MATERIALS AND METHODS: A group of 14 patients with high-grade ICA stenosis and 11 healthy control subjects were examined with 2D (1)H MRSI and 3D (31)P MRSI at 3 T before and after treatment of severe ICA stenosis. Spectroscopic data were processed with LCModel and jMRUI software. Changes of the phosphorylated metabolites, pH, N-acetyl-acetate, creatine and choline-containing compounds prior/post intervention were analyzed and patients' data were compared with that of control subjects. RESULTS: Untreated patients had significantly higher Adenosindiphosphate (ADP) in basal ganglia ipsi- and contralateral to the side of ACI stenosis compared to controls. After treatment, ADP of both hemispheres significantly decreased by approximately 20% compared to the pre-treatment values. Further, significant decreases of phosphorylated metabolites prior/post intervention were found for patients compared to controls. CONCLUSION: This spectroscopic study reveals that unilateral high-grade ICA stenosis has an effect on cerebral high-energy metabolism of both hemispheres, which is at least partially reversible after treatment. Therefore the restoration of blood flow in high-grade ICA stenosis recovers the impaired energy balance of the brain.

Diagnostic impact of proton MR-spectroscopy versus image-guided stereotactic biopsy.

BACKGROUND: The aim of this study was to compare the diagnostic accuracy of (1)H MR-spectroscopy versus image-guided stereotactic biopsy. METHOD: A cohort of 83 consecutive patients with a broad spectrum of brain lesions were examined. Prior to stereotactic biopsy, the patients were subjected to (1)H MR-spectroscopy examination. Diagnostic accuracy of (1)H MR-spectroscopy and image guided stereotactic biopsy was determined for the largest diagnostic subgroups. Each diagnostic procedure was tested for concordance in every subgroup. FINDINGS: The subgroups of patients comprised: low grade glioma, high grade glioma (grades III and IV), lymphoma and metastasis. For the sensitivity of (1)H MR-spectroscopy ranged from 87.7 in high grade glioma to 92.3% in metastasis and for specificity from 93.3% for high grade glioma to 100% in low grade glioma. The highest positive predictive value of 100% was reached in the subgroup of low grade glioma. The highest negative predictive value was reached in lymphoma and metastasis, 100%. The kappa values were highly significant for all comparisons (p<0.001). The co-efficient ranged from 0.68 to 0.84. It was lowest in assessing high grade glioma and highest in lymphoma. CONCLUSION: Compared with each other (1)H MR-spectroscopy and image-guided stereotactic biopsy showed a moderate to good, statistically highly significant concordance. In patients in whom operation is at an increased risk e.g., due to severe medical illness, (1)H MR-spectroscopy as a noninvasive procedure may be sufficient to assess the diagnosis.

Proton magnetic resonance spectroscopy in childhood brainstem lesions.

BACKGROUND: Diagnosis of brainstem lesions in children based on magnetic resonance imaging alone is a challenging problem. Magnetic resonance spectroscopy (MRS) is a noninvasive technique for spatial characterization of biochemical markers in tissues and gives information regarding cell membrane proliferation, neuronal damage, and energy metabolism. METHODS: We measured the concentrations of biochemical markers in five children with brainstem lesions and evaluated their potential diagnostic significance. Images and spectra were acquired on a 1.5-T imager. The concentrations of N-acetylaspartate, tetramethylamines (e.g., choline), creatine, phosphocreatine, lactate, and lipids were measured within lesions located at the brainstem using Point-resolved spectroscopy sequences. RESULTS: Diagnosis based on localized proton spectroscopy included brainstem glioma, brainstem encephalitis, demyelination, dysmyelination secondary to neurofibromatosis type 1 (NF 1), and possible infection or radiation necrosis. In all but one patient, diagnosis was confirmed by biopsy or by clinical follow-up. CONCLUSIONS: This small sample of patients suggests that MRS is important in the differential diagnosis between proliferative and nonproliferative lesions in patients without neurofibromatosis. Unfortunately, in cases of NF 1, MRS can have a rather misdiagnosis role.

An unusual intraventricular haemangiopericytoma: MRI and spectroscopy.

We present a 43-year-old woman with a tumour within the left lateral ventricle with the typical appearances of meningioma on MRI. (1)H MR spectroscopy demonstrated an increased choline peak, suggesting a malignant form of meningioma. Histologically a haemangiopericytoma was found, an exceptionally rare tumour at this site.

Proton MR spectroscopy of neurometabolites in hepatic encephalopathy during L-ornithine-L-aspartate treatment--results of a pilot study.

Hepatic encephalopathy (HE) is associated with typical changes in neurometabolites most likely being caused by an elevated systemic ammonia level. Blood ammonia is a valid over-all biomarker of HE and thus is regularly determined in clinical trials. The neurometabolites affected in HE can be assessed in vivo by proton magnetic spectroscopy. The aim of this study was to show the effect of the ammonia lowering drug L-ornithine-L-aspartate (OA) on the cerebral glutamate+glutamine/creatine (Glu+GLN/Cr) ratio. In an open clinical trial (pilot study), 15 patients with stable HE were treated with an infusion of 40 g OA over 8 h (5 g/h). Immediately before and 6-8 (mean 6.8) h after start of the infusion, spectroscopy of the parietal white matter was performed and arterial blood ammonia quantified. Glu+GLN/Cr-ratios correlated significantly with ammonia data (Spearman's correlation coefficient rs = 0.72, p < 0.001). Likewise, the OA induced changes (versus baseline before infusion) in blood ammonia level and in Glu+GLN/Cr-ratios correlated significantly rs = 0.54, p = 0.0375). Magnetic resonance (MR) spectroscopy assesses the neurometabolite variation present in HE. OA induced changes in cerebral Glu+GLN/ Cr-ratio were significantly correlated with the drug effects on arterial blood ammonia. These pilot data indicate that MR spectroscopy detects a specific biomarker of HE that may reflect the extent of the cerebral alterations associated with the disease.

Clinical application of proton magnetic resonance spectroscopy in the diagnosis of intracranial mass lesions.

Diagnosis of primary and secondary brain tumours and other focal intracranial mass lesions based on imaging procedures alone is still a challenging problem. Proton magnetic resonance spectroscopy (1H-MRS) gives completely different information related to cell membrane proliferation, neuronal damage, energy metabolism and necrotic transformation of brain or tumour tissues. Our purpose was to evaluate the clinical utility of 1H-MRS added to MRI for the differentiation of intracranial neoplastic and non-neoplastic mass lesions. 176 mostly histologically verified lesions were studied with a constant clinically available single volume 1H-MRS protocol following routine MRI. 12 spectra (6.8%) were not of satisfactory diagnostic quality; 164 spectroscopic data sets were therefore available for definitive evaluation. Our study shows that spectroscopy added to MRI helps in tissue characterization of intracranial mass lesions, thereby leading to an improved diagnosis of focal brain disease. Non-neoplastic lesions such as cerebral infarctions and brain abscesses are marked by decreases in choline (Cho), creatine (Cr) and N-acetyl-aspartate (NAA), while tumours generally have elevated Cho and decreased levels of Cr and NAA. Gliomas exhibit significantly increased Cho and lipid formation with higher WHO tumour grading. Metastases have elevated Cho similar to anaplastic astrocytomas, but can be differentiated from high-grade gliomas by their higher lipid levels. Extra-axial tumours, i.e. meningiomas and neurinomas, are characterized by a nearly complete absence of the neuronal marker NAA. The additive information of 1H-MRS led to a 15.4%-higher number of correct diagnoses, to 6.2% fewer incorrect and 16% fewer equivocal diagnoses than with structural MRI data alone.

Real-time measurements of cellular oxygen consumption, pH, and energy metabolism using nuclear magnetic resonance spectroscopy.

Changes in molecular expression or apoptotic behavior, induced by malignant transformation or anticancer treatment, are frequently reflected in cellular metabolism and oxygen consumption. A technique to monitor oxygen consumption, cell physiology, and metabolism noninvasively would provide a better understanding of interactions between molecular changes and metabolism in malignant transformation and following cancer treatment. Such a system was developed in this study by adapting multinuclear MRI and spectroscopic techniques to an isolated cell perfusion system. The system was evaluated by studying the effects of two agents, carbonyl cyanide m-chlorophenylhydrazone (CCCP) which is an uncoupler of oxidative phosphorylation, and antimycin, an inhibitor of oxidative phosphorylation, on the oxygen consumption and metabolism of MCF-7 and MatLyLu cancer cell lines.

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells.

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.

Imaging prostate cancer invasion with multi-nuclear magnetic resonance methods: the Metabolic Boyden Chamber.

The physiological milieu within solid tumors can influence invasion and metastasis. To determine the impact of the physiological environment and cellular metabolism on cancer cell invasion, it is necessary to measure invasion during well-controlled modulation of the physiological environment. Recently, we demonstrated that magnetic resonance imaging can be used to monitor cancer cell invasion into a Matrigel layer [Artemov D, Pilatus U, Chou S, Mori N, Nelson JB, and Bhujwalla ZM (1999). Dynamics of prostate cancer cell invasion studied in vitro by NMR microscopy. Mag Res Med 42, 277-282.]. Here we have developed an invasion assay ("Metabolic Boyden Chamber") that combines this capability with the properties of our isolated cell perfusion system. Long-term experiments can be performed to determine invasion as well as cellular metabolism under controlled environmental conditions. To characterize the assay, we performed experiments with prostate cancer cell lines preselected for different invasive characteristics. The results showed invasion into, and degradation of the Matrigel layer, by the highly invasive/metastatic line (MatLyLu), whereas no significant changes were observed for the less invasive/metastatic cell line (DU-145). With this assay, invasion and metabolism was measured dynamically, together with oxygen tensions within the cellular environment and within the Matrigel layer. Such a system can be used to identify physiological and metabolic characteristics that promote invasion, and evaluate therapeutic interventions to inhibit invasion.

Dynamics of prostate cancer cell invasion studied in vitro by NMR microscopy.

Understanding the dynamics and pathogenesis of invasion is vital for developing strategies to prevent cancer metastasis. Conventional invasion assays provide information for a single time point. NMR microscopic imaging as used in the current study to measure cell invasion in vitro provides a nondestructive method for scoring cell invasion thus offering a unique possibility to study this process dynamically. An additional advantage is that cells can be retrieved for metabolic and physiological characterization. Two prostate cancer cell lines, DU-145 and Mat-Ly-Lu, preselected for differences in invasive behavior, were studied. Cells were seeded in 12-mm culture plate inserts containing a 15-microm-thick porous membrane with 3.0 microm pore size that was coated with a 100 microm Matrigel layer. Cell invasion in the Matrigel layer was obtained from the profile of intracellular water measured with diffusion-weighted 1D imaging. Additional experiments were also performed with confocal microscopy to validate the NMR results. Significant differences were detected between the invasive behavior of DU-145 and Mat-Ly-Lu cells. The obtained results show that NMR microscopy can be used to dynamically study invasion by cancer cells. The noninvasive nature of NMR microscopy permits determination of cell migration dynamically for any given sample, which is especially important if cell availability is limited to the unique sample, such as for biopsy specimens. Magn Reson Med 42:277-282, 1999.

Characterization of a major permeability barrier in the zebrafish embryo.

Fish embryos represent a class of multicompartmental biological systems that have not been successfully cryopreserved, primarily because of the lack of understanding of how water and cryoprotectants permeate the compartments. We are using the zebrafish embryo as a model to understand these kinetics. Zebrafish embryos have two major compartments, the blastoderm and the yolk, which is surrounded by the multinucleated yolk syncytial layer (YSL). We determined the water and cryoprotectant permeability in these compartments using two methods. First, we measured shrink/swell dynamics in optical volumetric experiments. Zebrafish embryos shrank over time and did not re-expand while immersed in dimethyl sulfoxide (DMSO) or propylene glycol. Second, we measured DMSO uptake with diffusion-weighted nuclear magnetic resonance spectroscopy. DMSO uptake was rapid during the first few minutes, then gradual thereafter. We used one- and two-compartment models to analyze the data and to determine the permeability parameters. We found that the two-compartment model provided a better fit to the data. On the basis of this model and in the presence of DMSO, the yolk and blastoderm had very similar water permeabilities (i.e., 0.01 and 0. 005 micron x min-1atm-1, respectively), but they had different DMSO permeabilities separated by three orders of magnitude (i.e.,