Geirmund Unsgård

SonoWand, an Ultrasound-based Neuronavigation System

OBJECTIVEWe have integrated a neuronavigation system into an ultrasound scanner and developed a single-rack system that enables the surgeon to perform frameless and armless stereotactic neuronavigation using intraoperative three-dimensional ultrasound data as well as preoperative magnetic resonance or computed tomographic images. The purpose of this article is to describe our two-rack prototype and present the results of our work on image quality enhancement. DESCRIPTION OF INSTRUMENTATIONThe system consists of a high-end ultrasound scanner, a modest-cost computer, and an optical positioning/digitizer system. Special technical and clinical efforts have been made to achieve high image quality. A special interface between the ultrasound instrument and the navigation computer ensures rapid transfer of digital three-dimensional data with no loss of image quality. OPERATIVE TECHNIQUEThe positioning system tracks the position and orientation of the patient, the ultrasound probe, the pointer, and various surgical instruments. This makes it possible to update the three-dimensional map during surgery and navigate by ultrasound data in a similar manner as with magnetic resonance data. METHODSThe two-rack prototype has been used for clinical testing since November 1997 at the University Hospital in Trondheim. EXPERIENCE AND RESULTSThe image quality improvements have enabled us, in most cases, to extract information from ultrasound with clinical value similar to that of preoperative magnetic resonance imaging. The overall clinical accuracy of the ultrasound-based navigation system is expected to be comparable to or better than that of a magnetic resonance imaging-based system. CONCLUSIONThe SonoWand system enables neuronavigation through direct use of intraoperative three-dimensional ultrasound. Further research will be necessary to explore the potential clinical value and the limitations of this technology.

Direct demonstration by [13C]NMR spectroscopy that glutamine from astrocytes is a precursor for GABA synthesis in neurons.

Primary cultures of cerebral cortical astrocytes and neurons, as well as neurons growing on top of the astrocytes (sandwich co-cultures), were incubated with 1-[13C]glucose or 2-[13C]acetate and in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. [13C]NMR spectroscopy at 125 MHz was performed on perchloric acid extracts of the cells or on media collected from the cultures. In addition, the [13C/12C] ratios of the amino acids glutamine, glutamate and 4-aminobutyrate (GABA) were determined by gas chromatography/mass spectroscopy, showing a larger degree of labeling in GABA than in glutamate and glutamine from glucose. Glutamine and glutamate were predominantly labeled from acetate. A picture of cellular metabolism mainly regarding the tricarboxylic acid cycle and glycolysis was obtained. Due to the fact that acetate is not metabolized by neurons to any significant extent, it could be shown that precursors from astrocytes are incorporated into the GABA pool of neurons grown in co-culture with astrocytes. Spectra of media removed from these cultures revealed that likely precursor candidates for GABA were glutamine and citrate. The importance of glutamine is further substantiated by the finding that inhibition of glutamine synthetase, an enzyme present in astrocytes only, significantly decreased the labeling of GABA in co-cultures incubated with 2-[13C]acetate.

Metabolism of [U-13C]glutamate in astrocytes studied by 13C NMR spectroscopy: incorporation of more label into lactate than into glutamine demonstrates the importance of the tricarboxylic acid cycle.

Abstract: Primary cultures of cerebral cortical astrocytes were incubated with [U‐13C]glutamate (0.5 mM) in modified Dulbeccos medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify 13C‐labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C‐3 position and two doublets in the C‐2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton‐decoupled 13C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C‐3 position not only showed the expected apparent triplet but also a doublet due to 13C incorporation into the C‐4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [13C]glutamate in the PCA extracts showed that 19% of the glutamate contained 12C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of 12C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C‐3 resonances of these compounds. Only a maximum of 30% was converted to glutamine directly.

Preoperative blood oxygen level-dependent functional magnetic resonance imaging in patients with primary brain tumors: clinical application and outcome.

OBJECTIVEThis study sought to evaluate the ability of blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to successfully identify functional cortical areas in patients with primary brain tumors, to evaluate the use of the fMRI results in presurgical planning, and to assess the functional outcome of the patients with respect to the functional maps obtained with fMRI. METHODSThe study included 25 consecutive preoperative fMRI sessions in patients with primary brain tumors in or near sensorimotor and/or language cortices. All fMRI paradigms were analyzed and rated according to the degree of success. Several distances between tumor and functional cortex as delineated with BOLD fMRI were measured to assess the topographic relationship between these two structures. Pre- and postoperative neurological statuses were obtained from the patients’ journals. RESULTSAcquisition of BOLD fMRI images was successful in 80% of the cases. The primary cause of unsuccessful fMRI was echo-planar imaging signal voids that were the result of previous craniotomy; the secondary cause was excessive motion. The neurosurgeons used the fMRI results for preoperative planning in 75% of the cases in which fMRI was successful. The risk of postoperative loss of function tested with fMRI was significantly lower when the distance between tumor periphery and BOLD activity was 10 mm or more. CONCLUSIONThe majority of patients with primary brain tumors were capable of satisfactorily performing the fMRI paradigms, and the information obtained was used in the preoperative planning. A distance of 10 mm or more between the functional cortex, as delineated with fMRI, and the tumor significantly reduced the risk of postoperative loss of function.

First direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes.

Primary cultures of cerebral cortical neurons or astrocytes or the two cell types together (co-cultures) were incubated with [1-13C]glucose for 20 or 48 h. Subsequently, perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy in order to detect 13C-labeled amino acids (glutamine, glutamate, gamma-aminobutyrate (GABA)) and other metabolites (lactate, tricarboxylic acid cycle (TCA) constituents). NMR spectra of PCA extracts of neurons or co-cultures exhibited distinct peaks for glutamate and GABA whereas the PCA extracts of astrocytes and co-cultures showed peaks corresponding to glutamine and glutamate. This pattern is consistent with the neuronal location of the GABA synthesizing enzyme glutamate decarboxylase and the astrocytic localization of the glutamine synthesizing enzyme, glutamine synthetase. NMR spectra of the incubation media showed clearly that 13C-labeled citrate, alanine and glutamine were synthesized and released from astrocytes since only media from the astrocyte cultures or co-cultures or neurons and astrocytes contained these metabolites in detectable amounts. It may be concluded that astrocytes play an important role supplying neurons with precursors for biosynthesis of glutamate and GABA such as glutamine and TCA cycle constituents. Since among the latter only citrate could be found in significant amounts it may be hypothesized that this may be the quantitatively most important TCA constituent to be released from astrocytes and subsequently utilized by neurons.

13C MR Spectroscopy Study of Lactate as Substrate for Rat Brain

In order to address the question whether lactate in blood can serve as a precursor for cerebral metabolites, fully awake rats were injected intravenously with [U-13C]lactate or [U-13C]glucose followed 15 min later by decapitation. Incorporation of label from [U-13C]glucose was seen mainly in glutamate, GABA, glutamine, aspartate, alanine and lactate. More label was found in glutamate than glutamine, underscoring the predominantly neuronal metabolism of pyruvate from [U-13C]glucose. It was estimated that the neuronal metabolism of acetyl CoA from glucose accounts for at least 66% and the glial for no more than 34% of the total glucose consumption. When [U-13C]lactate was the precursor, label incorporation was similar to that observed from [U-13C]glucose, but much reduced. Plasma analysis revealed the presence of approximately equal amounts of [1,2,3-13C]- and [1,2-13C]glucose, showing gluconeogenesis from [U-13C]lactate. It was thus possible that the labeling seen in the cerebral amino acids originated from labeled glucose, not [U-13C]lactate. However, the presence of significantly more label in [U-13C]- than in [2,3-13C]alanine demonstrated that [U-13C]lactate did indeed cross the blood-brain barrier, and was metabolized further in the brain. Furthermore, contributions from pyruvate carboxylase (glial enzyme) were detectable in glutamine, glutamate and GABA, and were comparatively more pronounced in the glucose group. This indicated that relatively more pyruvate from lactate than glucose was metabolized in neurons. Surprisingly, the same amount of lactate was synthesized via the tricarboxylic acid cycle in both groups, indicating transfer of neurotransmitters from the neuronal to the astrocytic compartment, as previous studies have shown that this lactate is synthesized primarily in astrocytes. Taking into consideration that astrocytes take up glutamate more avidly than GABA, it is conceivable that neuronal lactate metabolism was more prominent in glutamatergic neurons.

NMR Spectroscopic Studies of 13C Acetate and 13C Glucose Metabolism in Neocortical Astrocytes: Evidence for Mitochondrial Heterogeneity

Neocortical astrocytes were incubated with 13C-labeled substrates to determine metabolic pathways. 13C NMR spectroscopy was used to analyze 13C incorporation into glutamine and citrate from the different precursors--[1-13C]glucose or [2-13C]acetate. When glucose was the labeling substrate, incorporation due to pyruvate carboxylation should be observed in the C-2 position in glutamine and the C-4 position in citrate. A large incorporation due to pyruvate carboxylation was observed in glutamine in the C-2 and C-3 positions, but not in citrate. When acetate was the precursor, the labeling ratios in the C-2/C-4 positions in glutamine and in the equivalent positions in citrate were 0.27 and 0.11, respectively. Moreover, acetate labeled lactate in the C-2 position much less than did glucose. Altogether, these observations led to the conclusion that glutamine precursors and citrate are either produced in different types of astrocytes or in different tricarboxylic acid cycles, situated in functionally different mitochondria in the same cell, and that in all likelihood pyruvate carboxylase is expressed differently in these mitochondria.

Combined Perfusion and Diffusion-Weighted Magnetic Resonance Imaging in a Rat Model of Reversible Middle Cerebral Artery Occlusion

BACKGROUND AND PURPOSE Diffusion-weighted imaging and dynamic first-pass bolus tracking of susceptibility contrast agents (perfusion imaging) are two new magnetic resonance imaging techniques that offer the possibility of early diagnosis of stroke. The present study was performed to evaluate the diagnostic information derived from these two methods in a rat model of temporary focal ischemia. METHODS Fifteen male Wistar rats were assigned to 45 (n = 7) or 120 minutes (n = 8) of middle cerebral artery occlusion followed by reperfusion using the intraluminal filament technique. The diffusion-weighted images were collected, and areas of hyperintensity were compared with histologically assessed areas of ischemic injury. The magnetic resonance perfusion image series were postprocessed to produce topographic maps reflecting the maximum reduction in the signal obtained during the first passage of the contrast agent and the time delay between the arrival of the bolus and the point of maximum contrast-agent effect. RESULTS Hyperintensity in diffusion-weighted images was demonstrated after 30 minutes of middle cerebral artery occlusion and was mainly expressed in the lateral caudoputamen and parts of the lower frontoparietal cortex. Reperfusion after 45 minutes of occlusion reduced the area of hyperintensity from 24.2% to 9.9% of hemispheric area. In the group with 120 minutes of occlusion, the hyperintense area increased from 24.4% to 29.1%. Relative to the nonischemic hemisphere, the changes in the topographic maps of maximum signal reduction occurred in the lateral caudoputamen and adjacent lower neocortical areas. Increased time delay to maximum effect, however, was seen also in the upper frontoparietal cortex. CONCLUSIONS Hyperintensity in diffusion-weighted images was reversible after 45 minutes but not after 120 minutes of middle cerebral artery occlusion. Analysis of the signal-reduction and time-delay parametric maps demonstrated regions of different perfusion changes in the ischemic hemisphere.

In vivo injection of [1-13C]glucose and [1,2-13C]acetate combined with ex vivo 13C nuclear magnetic resonance spectroscopy: a novel approach to the study of middle cerebral artery occlusion in the rat.

Astrocytes play a pivotal role in cerebral glutamate homeostasis. After 90 minutes of middle cerebral artery occlusion in the rat, the changes induced in neuronal and astrocytic metabolism and in the neuronal—astrocytic interactions were studied by combining in vivo injection of [1-13C]glucose and [1,2-13C]acetate with ex vivo 13C nuclear magnetic resonance spectroscopy and HPLC analysis of amino acids of the lateral caudoputamen and lower parietal cortex, representing the putative ischemic core, and the upper frontoparietal cortex, corresponding to the putative penumbra. In the putative ischemic core, evidence of compromised de novo glutamate synthesis located specifically in the glutamatergic neurons was detected, and a larger proportion of glutamate was derived from astrocytic glutamine. In the same region, pyruvate carboxylase activity, representing the anaplerotic pathway in the brain and exclusively located in astrocytes, was abolished. However, astrocytic glutamate uptake and conversion to glutamine took place, and cycling of intermediates in the astrocytic tricarboxylic acid cycle was elevated. In the putative penumbra, glutamate synthesis was improved compared with the ischemic core, the difference appeared to be brought on by better neuronal de novo glutamate synthesis, combined with normal levels of glutamate formed from astrocytic glutamine. In both ischemic regions, γ-aminobutyric acid synthesis directly from glucose was reduced to about half, indicating impaired pyruvate dehydrogenase activity; still, γ-aminobutyric acid reuptake and cycling was increased. The results obtained in the current study demonstrate that by combining in vivo injection of [1-13C]glucose and [1,2-13C]acetate with ex vivo 13C nuclear magnetic resonance spectroscopy, specific metabolic alterations in small regions within the rat brain suffering a focal ischemic lesion can be studied.

Differences in Neurotransmitter Synthesis and Intermediary Metabolism between Glutamatergic and GABAergic Neurons during 4 Hours of Middle Cerebral Artery Occlusion in the Rat: The Role of Astrocytes in Neuronal Survival

Astrocytes are intimately involved in both glutamate and γ-aminobutric acid (GABA) synthesis, and ischemia-induced disruption of normal neuroastrocytic interactions may have important implications for neuronal survival. The effects of middle cerebral artery occlusion (MCAO) on neuronal and astrocytic intermediary metabolism were studied in rats 30, 60, 120, and 240 minutes after MCAO using in vivo injection of [1-13C]glucose and [1,2-13C]acetate combined with ex vivo13C magnetic resonance spectroscopy and high-performance liquid chromatography analysis of the ischemic core (lateral caudoputamen and lower parietal cortex) and penumbra (upper frontoparietal cortex). In the ischemic core, both neuronal and astrocytic metabolism were impaired from 30 minutes MCAO. There was a continuous loss of glutamate from glutamatergic neurons that was not replaced as neuronal glucose metabolism and use of astrocytic precursors gradually declined. In GABAergic neurons astrocytic precursors were not used in GABA synthesis at any time after MCAO, and neuronal glucose metabolism and GABA-shunt activity declined with time. No flux through the tricarboxylic acid cycle was found in GABAergic neurons at 240 minutes MCAO, indicating neuronal death. In the penumbra, the neurotransmitter pool of glutamate coming from astrocytic glutamine was preserved while neuronal metabolism progressively declined, implying that glutamine contributed significantly to glutamate excitotoxicity. In GABAergic neurons, astrocytic precursors were used to a limited extent during the initial 120 minutes, and tricarboxylic acid cycle activity was continued for 240 minutes. The present study showed the paradoxical role that astrocytes play in neuronal survival in ischemia, and changes in the use of astrocytic precursors appeared to contribute significantly to neuronal death, albeit through different mechanisms in glutamatergic and GABAergic neurons.