Randy L. Tyson

Labeling of N-acetylaspartate and N-acetylaspartylglutamate in rat neocortex, hippocampus and cerebellum from [1-13C]glucose

Both N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) are localized almost exclusively to neurons, and have become important markers of neuronal viability in a number of cerebral pathological conditions. Using nuclear magnetic resonance spectroscopy combined with [1-13C]glucose administration (200 min infusion) we show that the synthesis of both NAA and NAAG can be observed. Label was incorporated into NAA from labeled acetate and from labeled aspartate, while NAAG was labeled from labeled glutamate. The low fractional enrichment of NAA (ca. 3%) relative to aspartate (20%) suggests a slow turnover rate, while NAAG (20.0%) and glutamate (25.2%) labeling were nearly equal, suggesting that NAAG labeling is near steady state. The rapid turnover of NAAG suggests an important role in glutamate delivery, while the slow rate of NAA turnover implies that its major role is as substrate for the formation of NAAG.

13C-Labeled substrates and the cerebral metabolic compartmentalization of acetate and lactate.

[1-13C]Glucose, [2-13C]acetate and [3-13C]lactate were infused into male Sprague-Dawley rats (150-170 g) for periods of 3-100 min (n=4 per time) and neocortex extracts were analyzed using 13C-edited 1H magnetic resonance (MR) spectroscopy. The time dependence of the [4-13C]glutamine/[4-13C]glutamate labeling ratio was significantly different for all three substrates infused (p<0.001) and showed that acetate is primarily utilized by glia and lactate by neurons, whereas glucose is ubiquitous. The ratio of second- to first-turn TCA cycle labeling for glutamine was significantly lower for acetate (30-100 min infusion; p<0.02) and greater for lactate (10-30 min; p<0.02) than for glucose infusions, while the C-2/C-4 glutamate labeling ratio was similar for all the three substrates. This indicated that transfer of [2-13C]acetate-derived [4-13C]glutamine to neurons was preferred to reentry of label into the glial TCA cycle and that the neuronal TCA cycle turnover is significantly faster than that for glia. Fitting parameters of a function representing a pseudo-first-order process to the time dependence of labeling demonstrated that GABA labeling reaches steady state faster with glutamine labeled from [2-13C]acetate than with glutamate labeled from [3-13C]lactate. It is concluded that lactate represents a significant improvement over glucose in the study of neuronal metabolism and complements the use of acetate to study glial metabolism and glial/neuronal metabolic relationships.

HUMAN CEREBRAL NEOPLASMS STUDIED USING MR SPECTROSCOPY : A REVIEW

Of primary central nervous system tumors treated each year, the majority are glioma, followed by meningioma and then pituitary adenoma. While the use of magnetic resonance (MR) and computed tomographic imaging is well established in the diagnosis and management of such tumors, these techniques have a limited role in determining the metabolic state, either prior to or following therapy. Multinuclear MR spectroscopy, on the other hand, provides information on tumor metabolism and the effect of therapy on tumor viability. This paper reviews MR spectroscopic studies performed on patients with central nervous system tumors and discusses the impact that such studies have had on tumor diagnosis and management.

Truncation of the Krebs Cycle During Hypoglycemic Coma

There is a misconception that hypoglycemic nerve cell death occurs easily, and can happen in the absence of coma. In fact, coma is the prerequisite for neuronal death, which occurs via metabolic excitatory amino acid release. The focus on nerve cell death does not explain how most brain neurons and all glia survive. Brain metabolism was interrogated in rats during and following recovery from 40 min of profound hypoglycemia using ex vivo (1)H MR spectroscopy to determine alterations accounting for survival of brain tissue. As previously shown, a time-dependent increase in aspartate was equaled by a reciprocal decrease in glutamate/glutamine. We here show that the kinetics of aspartate formation during the first 30 min (0.36 +/- 0.03 micromol g(-1) min(-1)) are altered such that glutamate, via aspartate aminotransferase, becomes the primary source of carbon when glucose-derived pyruvate is unavailable. Oxaloacetate is produced directly from alpha-ketoglutarate, so that reactions involving the six-carbon intermediates of the tricarboxylic acid cycle are bypassed. These fundamental observations in basic metabolic pathways in effect redraw the tricarboxylic acid cycle from a tricarboxylic to a dicarboxylic acid cycle during hypoglycemia. The basic neurochemical alterations according to the chemical equilibrium of mass action augments flux through a truncated Krebs cycle that continues to turn during hypoglycemic coma. This explains the partial preservation of energy charge and brain cell survival during periods of glucose deficiency.

Forebrain Ischemia in Diabetic and Nondiabetic BB Rats Studied With 31P Magnetic Resonance Spectroscopy

In spontaneously diabetic BB rats, the effect of chronically maintained blood glucose levels on the degree of energy failure and brain pH change during an ischemic insult, and on subsequent recovery after reperfusion, was studied with in vivo 31P magnetic resonance spectroscopy. Short duration forebrain ischemia (10-min carotid occlusion plus hypotension of 50 mmHg) was induced in diabetic and nondiabetic male BB rats whose blood glucose levels were maintained with insulin. Spectra were obtained in 1-min blocks before, during, and for 1 h after ischemia. Before ischemia, hypoglycemic (blood glucose <3 mM) diabetic rats had an increased PI peak intensity, with no significant pH change, compared with other groups. During ischemia, the rate and extent of hydrolysis of high-energy phosphate metabolites (as measured by an increase in PI) decreased, and the severity of tissue acidosis increased as preischemia blood glucose concentration increased. Among hyperglycemic BB rats, similar ischemia-induced changes were found for subgroups with blood glucose levels of 13.7 ± 1.2 and 20.3 ± 0.6 mM, in keeping with the known decrease in hexose binding sites associated with chronic hyperglycemia. Decline in PCr level during ischemia was not significantly different between groups. With reperfusion, both PI and pH values rapidly returned to preischemia values. PCr levels, however, did not recover in hyperglycemic diabetic animals, with the degree of residual impairment dependent on the preischemia glucose level. Results suggest that optimal management of diabetes may lessen the degree of injury within the ischemic penumbra in diabetic patients who suffer a stroke.

6-Aminonicotinamide inhibition of the pentose phosphate pathway in rat neocortex.

6-Aminonicotinamide (6-AN) is thought to inhibit the pentose phosphate pathway (PPP) since large increases in 6-phosphogluconate are observed following its administration. Immediately following 45 min i.v. infusion of [2-13C]glucose to controls and 6-AN-treated (50 mg/kg i.p. given 4 h previously) Sprague-Dawley rats (n = 5 for both groups), metabolism was arrested using freeze-funnel fixation. Chloroform-methanol-water neocortical extracts from animals administered with 6-AN demonstrated elevated levels of 6-phosphogluconate and 6-phosphoglucono-δ-lactone, both of which demonstrated labeling through metabolism of [2-13C]glucose. Comparison of the C-2 and C-3 lactate positions using 1H NMR spectroscopy showed that the fraction of glucose metabolized through the PPP is unchanged by 6-AN (14 ± 0.6% vs 14 ± 0.3% in control animals). It is hypothesized that as the PPP is inhibited by metabolites of 6-AN in the neocortex, glycolysis is inhibited in a proportionate manner through an inhibitory effect on phosphoglucose isomerase by 6-phosphogluconate and/or 6-phosphoglucono-δ-lactone.

Lactate Storm Marks Cerebral Metabolism following Brain Trauma

Background: In brain metabolism, neurons are fueled by lactate passed to them by glia in a metabolic coupling. Results: Following brain trauma, lactate uptake into neurons from glia was impaired, producing a metabolic lactate storm. Conclusion: Brain trauma results in neuronal-glial metabolic uncoupling, releasing free lactate. Significance: Inhibition of lactate production or its removal may be an important therapeutic strategy for brain trauma. Brain metabolism is thought to be maintained by neuronal-glial metabolic coupling. Glia take up glutamate from the synaptic cleft for conversion into glutamine, triggering glial glycolysis and lactate production. This lactate is shuttled into neurons and further metabolized. The origin and role of lactate in severe traumatic brain injury (TBI) remains controversial. Using a modified weight drop model of severe TBI and magnetic resonance (MR) spectroscopy with infusion of 13C-labeled glucose, lactate, and acetate, the present study investigated the possibility that neuronal-glial metabolism is uncoupled following severe TBI. Histopathology of the model showed severe brain injury with subarachnoid and hemorrhage together with glial cell activation and positive staining for Tau at 90 min post-trauma. High resolution MR spectroscopy of brain metabolites revealed significant labeling of lactate at C-3 and C-2 irrespective of the infused substrates. Increased 13C-labeled lactate in all study groups in the absence of ischemia implied activated astrocytic glycolysis and production of lactate with failure of neuronal uptake (i.e. a loss of glial sensing for glutamate). The early increase in extracellular lactate in severe TBI with the injured neurons rendered unable to pick it up probably contributes to a rapid progression toward irreversible injury and pan-necrosis. Hence, a method to detect and scavenge the excess extracellular lactate on site or early following severe TBI may be a potential primary therapeutic measure.

23Na Nuclear Magnetic Resonance Spectral Changes During and After Forebrain Ischemia in Hypoglycemic, Normoglycemic, and Hyperglycemic Rats

BACKGROUND AND PURPOSE The severity of brain injury in animal models of forebrain ischemia increases with blood glucose level. During ischemia, energy failure is slower and maintenance of ion gradients is prolonged as the level of glycemia increases. It is not clear how the level of glycemia influences recovery of ion homeostasis on reperfusion. It has been shown that changes in the intensity of the multiple-quantum 23Na nuclear magnetic resonance (NMR) signals reflect changes in intracellular Na+ levels. We have used 23Na NMR spectroscopy to evaluate the influence of the level of glycemia on changes in Na+ concentration during and after forebrain ischemia in rats. METHODS Single-quantum (SQ) and double-quantum (DQ) 23Na NMR spectra were measured before and during 10-minute forebrain ischemia and during reperfusion in hypoglycemic, normoglycemic, and hyperglycemic rats. RESULTS The DQ 23Na NMR signal increased to 210% of preischemia intensity in all rats, but a delay in this increase was observed in normoglycemic and hyperglycemic animals. The rate of the DQ 23Na NMR signal increase was fastest in hypoglycemic (apparent first-order rate constant 0.673 +/- 0.046 min-1, P < .002 compared with normoglycemic animals) and slowest in hyperglycemic (0.285 +/- 0.024 min-1, P < .03) rats. During reperfusion, the signal intensity recovered rapidly in hypoglycemic (0.385 +/- 0.050 min-1) and normoglycemic (0.464 +/- 0.047 min-1) rats, whereas in hyperglycemic animals recovery was slow (0.108 +/- 0.044 min-1, P < .0001 compared with normoglycemic animals). The SQ 23Na NMR signal intensity increased to 117% of preischemia level in hypoglycemic (P < .05 compared with normoglycemic animals) and to 107% in normoglycemic and hyperglycemic animals during reperfusion. CONCLUSIONS The slower increase in the 23Na DQ NMR signal intensity during forebrain ischemia in rats with higher blood glucose levels suggests that Na+ homeostasis is maintained longer in these animals. On reperfusion, the slower recovery of the DQ 23Na NMR signal intensity in hyperglycemic animals likely indicates a slower recovery of Na+ homeostasis, perhaps contributing to the increased neuronal injury after cerebral ischemia in hyperglycemic animals.

Δ9-Tetrahydrocannabinol increases brain temperature and inverts circadian rhythms

Δ9-tetrahydrocannabinol (THC) has been shown to protect against focal and global ischemia. Hypothermia is thought to be one mechanism for this protection. These observations are important since brain hyperthermia is known to increase ischemic damage while hypothermia is protective. To establish the effect of THC on brain and body core temperature, brain and body temperature probes were inserted for chronic temperature monitoring (n = 20). THC treated groups were administered THC at either low (0.1 mg/kg) or high (10 mg/kg) dose for 1 week. Brain temperature was recorded during this period and for 1 week following the discontinuation of THC. Chronic administration of THC at either dose increased brain temperature (p < 0.0001) but did not significantly change body core temperature (p = 0.4767) in the freely moving rat.

Molecular susceptibility weighted imaging of the glioma rim in a mouse model

BACKGROUND Glioma is the most common and most difficult to treat brain cancer. Despite many efforts treatment, efficacy remains low. As neurosurgical removal is the standard procedure for glioma, a method, allowing for both early detection and exact determination of the location, size and extent of the tumor, could improve a patients positive response to therapy. NEW METHOD We propose application of susceptibility weighted molecular magnetic resonance imaging using, targeted contrast agents, based on superparamagnetic iron oxide nanoparticles, for imaging of the, glioma rim, namely brain-tumor interface. Iron oxide attached to the targeted cells increases, susceptibility differences at the boundary between tumor and normal tissue, providing the opportunity, to utilize susceptibility weighted imaging for improved tumor delineation. We investigated potential, enhancement of the tumor-brain contrast, including tumor core and rim when using susceptibility, weighted MRI for molecular imaging of glioma. RESULTS There were significant differences in contrast-to-noise ratio before, 12 and 120min after contrast, agent injection between standard gradient echo pulse sequence and susceptibility weighted molecular, magnetic resonance imaging for the core-brain, tumor rim-core and tumor rim-brain areas. COMPARISON WITH EXISTING METHODS Currently, the most common MRI contrast agent used for glioma diagnosis is a non-specific, gadolinium-based agent providing T1-weighted enhancement. Susceptibility-weighted magnetic, resonance imaging is much less efficient when no targeted superparamagnetic contrast agents are, used. CONCLUSION The improved determination of glioma extent provided by SWI offers an important new tool for, diagnosis and surgical planning.