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, gamma-aminobutyric acid synthesis directly from glucose was reduced to about half, indicating impaired pyruvate dehydrogenase activity; still, gamma-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.     

Effects of repeated cerebral ischemia on extracellular amino acid concentrations measured with intracerebral microdialysis in the gerbil hippocampus

BACKGROUND AND PURPOSE: To clarify the role of elevated extracellular amino acid concentrations during ischemia on the cumulative neuronal damage after repeated cerebral ischemic insults, using a microdialysis technique we measured concentrations of the amino acids glutamate, glutamine, glycine, taurine, and gamma-aminobutyric acid in the gerbil hippocampus over three 2-minute forebrain ischemic insults induced at 1-hour intervals. METHODS: Under light anesthesia, the bilateral common carotid arteries were occluded with aneurysm clips at 1-hour intervals. Samples were collected by microdialysis at 10-minute intervals, and the amino acid concentrations were determined using a high-performance liquid chromatography system. RESULTS: During and immediately after the first ischemic insult, concentrations of glutamate, glycine, and taurine, but not glutamine, increased significantly. Glutamate and taurine concentrations rose again during the second and third ischemic insults, but the increases were smaller than those during the first insult. By contrast, glutamine concentrations increased slightly but significantly during the second and third ischemic insults. The extracellular concentration of gamma-aminobutyric acid before the ischemic insults was below the level of detectability but increased markedly during each ischemic insult, with similar declines in the amounts released during later insults. Concentrations of all amino acids returned to baseline after 10 minutes of reperfusion and remained at baseline until the subsequent ischemic insult was induced. CONCLUSIONS: It is well established that glutamate released during ischemia plays a crucial role in ischemia-induced neuronal death. However, the present results indicate that cumulative neuronal damage following sublethal ischemic insults is not caused by an exaggerated release of excitatory amino acids during subsequent ischemic insults but strongly suggest that increased intracellular reactions leading to cell death play a major role.     

Comparison of lactate and glucose metabolism in cultured neocortical neurons and astrocytes using 13C-NMR spectroscopy

In cerebral cortical neurons, synthesis of the tricarboxylic acid (TCA) cycle-derived amino acids, glutamate and aspartate as well as the neurotransmitter of these neurons, gamma-aminobutyrate (GABA), was studied incubating the cells in media containing 0.5 mM [U-13C]glucose in the absence or presence of glutamine (0.5 mM). Lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance (NMR) spectroscopy and HPLC. The present findings were compared to results previously obtained using 1.0 mM [U-13C]lactate as the labeled substrate for the neurons. Regardless of the amino acids studied, incubation periods of 1 and 4 h resulted in identical amounts of 13C incorporated. Furthermore, the metabolism of lactate was studied under analogous conditions in cultured cerebral cortical astrocytes. The incorporation of 13C from lactate into glutamate was much lower in the astrocytes than in the neurons. In cerebral cortical neurons the total amount of 13C in GABA, glutamate and aspartate was independent of the labeled substrate. The enrichment in glutamate and aspartate was, however, higher in neurons incubated with lactate. Thus, lactate appears to be equivalent to glucose with regard to its access to the TCA cycle and subsequent labeling of glutamate, aspartate and GABA. It should be noted, however, that incubation with lactate in place of glucose led to lower cellular contents of glutamate and aspartate. The presence of glutamine affected the metabolism of glucose and lactate differently, suggesting that the metabolism of these substrates may be compartmentalized.     

Quantification of the GABA shunt and the importance of the GABA shunt versus the 2-oxoglutarate dehydrogenase pathway in GABAergic neurons

We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2-oxoglutarate dehydrogenase for the conversion of 2-oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1-(13)C]glucose, and brain extracts were analyzed by 13C NMR spectroscopy. The percent enrichments of GABA C-2 and glutamate C-4 were the same: 5.0 +/- 1.6 and 5.1 +/- 0.2%, respectively (mean +/- SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the 13C labeling of aspartate from [1-(13)C]-glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [13C]aspartate during inhibition of the GABA shunt by gamma-vinyl-GABA indicated that not more than half the flux from 2-oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2-oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one-third of the overall cerebral TCA cycle activity in the mouse. Treatment with gamma-vinyl-GABA, which increased GABA levels dramatically, caused changes in the 13C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.     

Transmitter amino acid release from rat neocortex: complete versus incomplete ischemia models

Release of the excitotoxic amino acids, glutamate and aspartate, from the ischemic rat cerebral cortex was compared in two models; the seven vessel occlusion model (7VO) of complete cerebral ischemia and the four vessel occlusion model (4VO) of incomplete cerebral ischemia. Amino acid efflux into cortical superfusates was measured using cortical cups placed on both hemispheres. Whereas a 20 min period of ischemia causes a pronounced release of glutamate and aspartate from the 4VO model, efflux was significantly reduced in the 7VO model. Release of the inhibitory transmitter GABA, was similar in the two models. This result suggests that excitotoxic amino acid efflux into the extracellular spaces of the cerebral cortex may be enhanced by the residual blood flow in an incomplete ischemia.     

Induction of ischemic tolerance following brief focal ischemia in rat brain

The objective of this study was to determine whether brief focal ischemia induces ischemic tolerance in rat brain. Focal ischemia was produced in Wistar rats by occluding the middle cerebral artery (MCA) for 20 min at a distal site. Following recovery for 24 h, the animals were subjected to a 10-min episode of forebrain ischemia using a combination of bilateral carotid artery occlusion and systemic hypotension. Histologic injury, assessed after a survival period of 3-4 days, consisted of selective neuronal necrosis bilaterally in cerebral cortex, striatum, hippocampus, and thalamus superimposed upon a small cortical infarct adjacent to the site of MCA occlusion. However, the intensity of neuronal necrosis in the MCA territory of the neocortex ipsilateral to MCA occlusion was markedly less than that in the contralateral MCA cortex. In contrast, the extent of neuronal necrosis in subcortical structures was similar in both hemispheres. Unexpectedly, animals in which the MCA was manipulated, but not occluded, also exhibited a marked reduction of neuronal necrosis in the ipsilateral MCA neocortex following forebrain ischemia. However, in animals with craniotomy alone, forebrain ischemia caused a similar extent of neuronal necrosis in the MCA neocortex of both hemispheres. Transient occlusion of the MCA induced the focal expression of the 72-kDa heat-shock protein (hsp72) in the MCA territory of the neocortex. Limited expression of hsp72 was also detected following sham occlusion, but not after craniotomy alone. These results demonstrate focal induction of ischemic tolerance in rat neocortex that may be related to expression of heat-shock proteins.     

Gangliosides prevent reduction in exploratory activity and delayed neuronal death in gerbils subjected to transitory cerebral ischemia

INTRODUCTION: The ganglioside GM1 has been shown to be effective in the treatment of experimental cerebral ischemia. Gangliosides from bovine brain have not been used in the treatment of ischemic cerebral accidents. There is evidence suggesting that they may also be effective. RESULTS: Ten minutes of bilateral occlusion of the carotid arteries of Mongolian gerbils leads a week later to reduced spontaneous exploratory activity, assessed by counting the number of times they stood up in an open field over a period of three minutes, and retarded neuronal death in the pyramidal stratum of the CA1 sector of the hippocampus, evaluated on the density of normal neurons in this region of both hemispheres. Treatment with 30 mg/kg of intra-peritoneal bovine cerebral gangliosides during the first six days following occlusion of the carotid arteries, leads to conservation of both exploratory activity and density of pyramidal neurons observed in the control animals. CONCLUSIONS: Bovine cerebral gangliosides have a short term cytoprotector effect on neurons sensitive to the ischemia-reperfusion phenomenon. This effect may be due to more than one mechanism, in which other gangliosides (together with GM1) may be present due to transient permeability of the blood-brain barrier.     

Changes in levels of monoamines and their metabolites in incompletely ischemic brains of spontaneously hypertensive rats

In order to investigate changes in levels of monoamines and their related substances together with those of other neurotransmitters (acetylcholine and GABA), choline and substances related to energy metabolism (ATP, lactate and glucose) accompanying incomplete cerebral ischemia, a bilateral common carotid artery occlusion model of spontaneously hypertensive rats (SHR) was utilized. Animals were subjected to 1 or 2 h ischemia. Then the concentrations of substances were measured in the cerebral cortex, hippocampus and striatum and compared with control values. Due to the incomplete ischemia, ATP showed a moderate decrease, while lactate and choline increased remarkably, and GABA underwent a moderate increase. With regard to monoamines, both noradrenaline and serotonin levels were reduced in the cerebral cortex and hippocampus, whereas dopamine levels increased in the hippocampus. All monoamine metabolites, i.e. metabolites by monoamine oxidase (MAO), metabolites by catechol-O-methyltransferase (COMT), and metabolites by both MAO and COMT, underwent increases. The 3-methoxytyramine level in particular showed marked increases. Furthermore levels of precursor amino acids as well as 5-hydroxytryptophan rose. Acetylcholine decreased moderately only in the cerebral cortex. Among these changes, sustained increases in all the monoamine metabolites were characteristic of changes in the incompletely ischemic brain, suggesting that both COMT and MAO retain their activities in the incompletely ischemic brain.     

Cathepsin B and middle cerebral artery occlusion in the rat

Lysosomal proteases, although tightly regulated under physiological conditions, are known to contribute to cell injury after various forms of tissue ischemia have occurred. Because cathepsin B is a prominent lysosomal protease found in brain parenchyma, the authors hypothesized that it may contribute to neuronal cell death after focal cerebral ischemia. The authors measured the expression and spatial distribution of cathepsin B within the ischemic brain in 43 animals by means of immunohistochemical analysis in a rat model of transient middle cerebral artery (MCA) occlusion. Cathepsin B activity was also measured within specific ischemic brain regions by using an in vitro assay (22 animals). In addition, the authors tested the therapeutic effect of preischemic intraventricular administration of stefin A, a cysteine protease inhibitor, on the volume of cerebral infarction after transient MCA occlusion (15 animals). Increased cathepsin B immunoreactivity was detected exclusively within the ischemic neurons after 2 hours of reperfusion following a 2-hour MCA occlusion. Cathepsin B immunolocalization in the ischemic region decreased by 24 hours of reperfusion, but then increased by 48 hours of reperfusion because the infarct was infiltrated by inflammatory cells. Increased immunolocalization of cathepsin B in the inflammatory cells located in the necrotic infarct core continued through 7 days of reperfusion. Cathepsin B enzymatic activity was significantly increased in the ischemic tissue at 2, 8, and 48 hours, but not at 24 hours of reperfusion after 2 hours of MCA occlusion. Continuous intraventricular infusion of stefin A, before 2 hours of MCA occlusion (15 animals), significantly reduced infarct volume compared with control animals (12 animals): the percentage of hemispheric infarct volume was 20+/-3.9 compared with 33+/-3.5 (standard error of the mean; p = 0.025). These data indicate that neuronal cathepsin B undergoes increased expression and activation within 2 hours of reperfusion after a 2-hour MCA occlusion and may be a mechanism contributing to neuronal cell death. Intraventricular infusion of stefin A, an inhibitor of cathepsin B, significantly reduces cerebral infarct volume in rats.     

In vitro and ex vivo 13C-NMR spectroscopy studies of pyruvate recycling in brain

Pyruvate recycling is a well established pathway in the liver, but in the brain, the cellular localization of pyruvate recycling remains controversial and its physiological significance is unknown. In cultured cortical astrocytes, pyruvate formed from [U-13C]glutamate was shown to re-enter the TCA cycle after conversion to acetyl-CoA, as demonstrated by the labelling patterns in aspartate C-2 and C-3, lactate C-2, and glutamate C-4, which provides evidence for pyruvate recycling in astrocytes. This finding is in agreement with previous studies of astrocytic cultures, in which pyruvate recycling has been described from [U-13C]glutamine, in the presence of glutamate, and from [U-13C]aspartate. Pyruvate recycling in brain was studied in fasted rats receiving either an intraperitoneal or a subcutaneous injection of [1,2-13C]acetate followed by decapitation 30 min later. Extracts of cortical tissue were analysed with 13C-NMR spectroscopy and total amounts of amino acids quantified by HPLC. Plasma extracts were analysed with 1H- and 13C-NMR spectroscopy, and showed a significantly larger amount of [1, 2-13C]acetate in the intraperitoneal group compared to the subcutaneous group. Furthermore, a small amount of label was detected in glucose in both groups. In the subcutaneously injected rats, [4-13C]glutamate and [2-13C]GABA were less enriched than plasma glucose, which might have been the precursor. In the intraperitoneally injected rats, however, pyruvate formation from [1, 2-13C]acetate, and re-entry of this pyruvate into the TCA cycle was demonstrated by the presence of greater 13C enrichment in [4-13C]glutamate and [4-13C]glutamine compared to the subcutaneous group, probably resulting from the significantly higher [1, 2-13C]acetate concentration in brain and plasma.     

Portacaval anastomosis results in altered neuron--astrocytic metabolic trafficking of amino acids: evidence from 13C-NMR studies

13C-NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1-13C]glucose, whereas [2-13C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1-13C]glucose as precursor. Brain [1-13C]glucose metabolism in portacaval-shunted rats was similar to that in sham-operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The 13C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2-13C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval-shunted rats.     

Cerebral metabolic compartmentation as revealed by nuclear magnetic resonance analysis of D-[1-13C]glucose metabolism

Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, gamma-aminobutyrate (GABA), glutamine, and aspartate. D-[1-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for 13C-NMR analysis and amino acid analysis. The absolute amount of 13C present within each carbonatom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance 13C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo investigation of glutamate-glutamine metabolism in hyperammonemic monkey brain using 13C-magnetic resonance spectroscopy

To investigate the metabolism of glutamate and glutamine in living monkey brain, a system of in vivo 13C magnetic resonance spectroscopy (MRS) using 1H-decoupled 13C spectroscopy combined with monitoring temperature changes in the brain by MR phase mapping was developed. Serial 13C-NMR spectra of the amino acids glutamate and glutamine were acquired non-invasively over 4 h from anesthetized monkey brain after the intravenous administration of [1-13C]glucose (0.5-1.0 g/kg). In the acute hyperammonemic state induced by the administration of ammonium acetate (77 mg/kg bolus), it was observed that 13C incorporation into glutamine-4 was clearly accelerated, without changes of 13C incorporation into glutamate-4. During hyperammonemia, it was shown directly by [2-13C]glucose administration that the anaplerotic pathway for the TCA cycle was also augmented, contributing to the formation of glutamine in the astroglia.     

Prolongation and enhancement of postischemic c-fos expression after fasting

BACKGROUND AND PURPOSE: A rapid but transient expression of c-fos after cerebral ischemia has been extensively documented. However, the mechanism of this induction and whether induction of c-fos is neuroprotective or detrimental to the brain after ischemia is presently not clear. Fasting before transient cerebral ischemia has been shown to reduce delayed neuronal necrosis and infarct volume. The purpose of the present study was to examine the effect of preischemic fasting for 24 hours on the expression of c-fos after transient focal cerebral ischemia. METHODS: Focal cerebral ischemia was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries for 60 minutes. Male Long-Evans rats weighting 250 to 300 g were randomly divided into two groups: fed (control group) and food deprived for 24 hours (fasted group) before ischemic surgery. Infarct volumes were measured on the basis of triphenyltetrazolium chloride-delineated infarct areas, and plasma glucose levels were determined by the glucose oxidase method. Temporal and spatial expression of c-fos was assessed by Northern blot analysis, in situ hybridization, and immunohistochemistry. RESULTS: Fasting for 24 hours before 60 minutes of ischemia resulted in a 26.6% decrease in preischemic plasma glucose levels and a 74.5% reduction in infarct volumes in the fasted group compared with the control group. A rapid but transient induction of c-fos mRNA was observed in the ischemic cortex in control animals after 60 minutes of ischemia. Fasting not only prolonged but also enhanced the intensity of c-fos expression in the ischemic cortex. Regional c-fos expression was also different between these two groups. CONCLUSIONS: The results support the contention that c-fos expression may be compatible with its purported neuroprotective role in selected experimental paradigms. The signaling mechanisms underlying the effect of fasting and subsequent lowering of plasma glucose levels on postischemic c-fos expression remain to be explored.     

A study of the precision of confocal, ratiometric, Fura-2-based [Ca2+] measurements

Increased ammonia has been considered a key factor in the pathogenesis of hepatic encephalopathy. The high concentration of ammonia interferes with oxidative metabolism in the brain through an inhibitory effect on the tricarboxylic acid cycle (TCA). Inhibition of the TCA cycle may result in depletion of ATP. Due to the involvement of astrocytes in brain detoxification of ammonia, these cells are good candidates for studying ammonia's effect on energy stores in the brain. C6-glioma cells, which have altered glycolytic rates, may show greater sensitivity to the toxicity of ammonium chloride than astrocytes. To study the effect of ammonium chloride on energy storage of both astrocytes and C6-glioma, we observed the acute and chronic effects of NH4Cl (7.5 or 15 mM) on the metabolism of isolated astrocytes and C6-glioma cells. Primary astrocytes were isolated from the cerebral hemispheres of 1-2 day old Sprague-Dawley rats, and C6-glioma cells were purchased from the American Type Culture Collection (ATCC). Following treatment of the cells with ammonia, glucose, lactate, glutamate, ATP, and PCr were assayed. Our data showed that at 15 min following treatment with NH4Cl, there were no significant differences in the concentration of metabolites measured in astrocytes. However, following 15 min of treatment with NH4Cl, the concentration of some metabolites, for example, ATP and lactate, changed significantly in C6-glioma cells. We have shown that 24 h of treatment was sufficient time to see significant biochemical changes but not morphological changes in either cell type. Simultaneous biochemical and morphological changes were observed 48 h following treatment in C6-glioma cells and at 9-10 days following treatment in primary astrocytes. In primary astrocytes at 24 h following treatment, glucose utilization increased. This high utilization of glucose was in accordance with the increase in lactate and glutamate production and the decrease in ATP and PCr formation. In C6-glioma cells the utilization of glucose increased but this high utilization of glucose was consistent with a significant decrease in the concentration of lactate, glutamate and ATP.     

Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes

The metabolic fate of glutamate in astrocytes has been controversial since several studies reported > 80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U-13C] glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 mM [U-13C] glutamate 85% of the 13C metabolized was converted to glutamine. The formation of [1,2,3-13C3] glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2-0.5 mM glutamate, 13C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [13C] lactate was essentially unchanged within the range of 0.2-0.5 mM glutamate, whereas the amount of [13C] aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 mM, suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 mM and astrocytes from both rat and mouse brain are consistent with these findings.     

Compliance considerations with hormone replacement therapy

Lactate accumulation, amino acid aspartate and glutamate levels, and hypoxanthine, xanthine and malondialdehyde (MDA) concentrations were compared in neonate rat brain after transient global hypoxia induced alone or in association with unilateral ligation of a carotid artery. Lactate production in both hemispheres was higher in cerebral hypoxia-ischemia (CHI) than in cerebral hypoxia (CH), and was lower in CHI after 2 h than at 15 min of recovery. Aspartate and glutamate levels were reduced 15 min after CHI in both hemispheres, but aspartate alone was decreased 2 h after CHI in the ipsilateral (left) hemisphere and 15 min after CH in both hemispheres. Hypoxanthine was increased 15 min after CHI in the ipsilateral hemisphere but decreased at 2 h, whereas xanthine was increased. MDA production was not modified after CH or CHI. These data, compared to those obtained in adult animals suggest that glutamate release and the capacity to generate oxygen-derived radicals are lower in neonates after ischemia. These differences might explain why the brain of the mammalian neonate is much more resistant to CH and CHI than that of the adult.     

U1 snRNP inhibits pre-mRNA polyadenylation through a direct interaction between U1 70K and poly(A) polymerase

The brain damage produced by unilateral cerebral hypoxia-ischemia in the immature rat results from major alterations in cerebral energy metabolism and glucose utilization which begin during the course of the insult and proceed into the recovery period. Consistent with a lack of pathology, the alterations in the hemisphere contralateral to the carotid artery ligation are transient and return to normal within 24 h of recovery, whereas the hemisphere ipsilateral to the ligation exhibits both early and late responses, and infarction. The facilitative glucose transporter proteins mediate glucose transport across the blood-brain barrier (55 kDa GLUT1), and into neurons and glia (GLUT3 and 45 kDa GLUT1), and demonstrate both early and late responses to perinatal hypoxia-ischemia. This study employed in situ hybridization histochemistry to investigate the temporal and regional patterns of GLUT1 and GLUT3 gene expression following a severe (2.5 h) hypoxic-ischemic insult in the 7-day old rat brain. Enhanced GLUT1 mRNA expression was apparent in cerebral microvessels of both hemispheres and remained elevated in the ipsilateral hemisphere through 24 h of recovery, consistent with our previous observation of increased microvascular 55 kDa GLUT1 protein. The expression of the neuronal isoform, GLUT3, was enhanced in penumbral regions, such as piriform cortex and amygdala, but was rapidly reduced in the affected areas of cortex, hippocampus and thalamus, reflecting necrosis. The late response, observed at 72 h of recovery, was characterized by extensive necrosis in the ipsilateral hemisphere, loss of GLUT3 expression, and a gliotic reaction including increased GLUT1 in GFAP-positive astrocytes. This study demonstrates that cerebral hypoxia-ischemia in the immature rat produces both immediate-early and long-term effects on the glucose transporter proteins at the level of gene expression.     

Compartmentation of [14C]glutamate and [14C]glutamine oxidative metabolism in the rat hippocampus as determined by microdialysis

Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 +/- 18 and 2.1 +/- 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-14C]glutamate and [U-14C]glutamine were 408 +/- 8 and 387 +/- 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

In vivo 13C NMR measurements of cerebral glutamine synthesis as evidence for glutamate-glutamine cycling

The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measured in rats in vivo under normal physiological and hyperammonemic conditions using 13C NMR spectroscopy. In the hyperammonemic animals, blood ammonia levels were raised from control values of approximately 0.05 mM to approximately 0.35 mM by an intravenous ammonium acetate infusion. Once a steady-state of cerebral metabolites was established, a [1-13C]glucose infusion was initiated, and 13C NMR spectra acquired continuously on a 7-tesla spectrometer to monitor 13C labeling of cerebral metabolites. The time courses of glutamate and glutamine C-4 labeling were fitted to a mathematical model to yield TCA cycle rate (V(TCA)) and the flux from glutamate to glutamine through the glutamine synthetase pathway (V(gln)). Under hyperammonemia the value of V(TCA) was 0.57 +/- 0.16 micromol/min per g (mean +/- SD, n = 6) and was not significantly different (unpaired t test; P > 0.10) from that measured in the control animals (0.46 +/- 0.12 micromol/min per g, n = 5). Therefore, the TCA cycle rate was not significantly altered by hyperammonemia. The measured rate of glutamine synthesis under hyperammonemia was 0.43 +/- 0.14 micromol/min per g (mean +/- SD, n = 6), which was significantly higher (unpaired t test; P < 0.01) than that measured in the control group (0.21 +/- 0.04 micromol/ min per g, n = 5). We propose that the majority of the glutamine synthetase flux under normal physiological conditions results from neurotransmitter substrate cycling between neurons and glia. Under hyperammonemia the observed increase in glutamine synthesis is comparable to the expected increase in ammonia transport into the brain and reported measurements of glutamine efflux under such conditions. Thus, under conditions of elevated plasma ammonia an increase in the rate of glutamine synthesis occurs as a means of ammonia detoxification, and this is superimposed on the constant rate of neurotransmitter cycling through glutamine synthetase.