George C. Newman

Can experimental conditions explain the discrepancy over glutamate stimulation of aerobic glycolysis

Uncertainty reigns over whether or not glutamate uptake in astrocytes leads to strong stimulation of glucose utilization, measured as accumulation of radioactive deoxyglucose-6-phosphate. This is an important issue, not only because glutamate is the major excitatory transmitter, but also because it has been postulated that glutamate-induced stimulation of glycolysis links brain excitation with activation of energy production. The effect of glutamate on deoxyglucose utilization in cultured rat and mouse astrocytes grown in different media and incubated under various conditions during the deoxyglucose assay has, therefore, been studied. Under most conditions, no stimulation occurred but rather a decrease in deoxyglucose utilization during exposure to glutamate; under certain conditions, the contribution of non-metabolized deoxyglucose to the intracellular 14C signal was significant.

Thick Brain Slices Model the Ischemic Penumbra

Hypothalamic brain slices, varying in thickness from 400μ to 1,000μ, were assessed by studying 2-deoxyglucose (2DG) metabolism, lactate accumulation, inulin spaces, and morphology at the light and ultrastructural levels. Evidence of increased glycolytic flux due to anaerobic metabolism is found at thicknesses greater than 600μ in association with a progressive increase in the inulin-exclusion space. The metabolic profiles, as a function of depth into the slices, reveal that 700-μ slices function in a manner similar to 540-μ slices at the surfaces, but with a core of increased 2DG phosphorylation at the slice center. In contrast, the 1000-μ slices show significant reductions of 2DG and increases in 2DG6P relative to the 540-μ slices at the slice surface as well as in the slice interior, suggesting impaired transport of 2DG into cells and spread of ischemic injury from the slice interior to the slice surface. Despite these metabolic changes, only minor morphologic changes of ischemic injury were found at the center of thicker slices, and in vitro glucose utilization of 1000-μ slices remained constant for up to 15 h. These three slice thicknesses should provide a useful model for studying the neurochemistry and neuropharmacology of the ischemic penumbra.

Effects of dextran on hippocampal brain slice water, extracellular space, calcium kinetics and histology

Hippocampal brain slices are valuable models for studying brain function but are compromised by several artifacts, including significant water gain and histologic injury, which occur under certain incubation conditions. Addition of colloid to Krebs-Ringer buffer (K-R) has been shown to eliminate water gain but has not achieved widespread acceptance. We confirm prior observations that dextran and PEG lessen the increase in slice mass during incubation in a dose-dependent manner with no water gain occurring at 4% concentrations. However, we also observe that addition of colloid to standard K-R induces severe neuronal pyknosis. Fortunately, the pyknosis can be eliminated by reduction in buffer osmolarity through adjustment of NaCl, producing markedly improved slice histology in dextran buffer, especially in the CA3 and CA4 regions of the hippocampus which are severely injured when incubated submerged in K-R at 37 degrees C. Extracellular space markers are not affected by either colloid. The volume of distribution for 45Ca is much larger in dextran buffers than in K-R and variability of 45Ca kinetics is also reduced. In the presence of dextran, hypoxia induces significant slice water gain, a relatively selective histologic injury and an alteration of tissue Ca2+ kinetics. Use of dextran buffers may eliminate many troubling brain slice artifacts.

Brain slice glucose utilization.

Abstract: The metabolism of 2‐deoxyglucose has been studied in 540 μm and 1,000 μm hypothalamic brain slices. Slice 2‐deoxyglucose (2DG) and 2‐deoxyglucose‐6‐phosphate (2DG6P) levels were measured after tissue homogenization and perchloric acid extraction. By analyzing the uptake and washout kinetics with nonlinear least‐squares methods, we have determined the rate constants for three‐, four‐, or five‐parameter kinetic models and obtained a value for the in vitro lumped constant (LC). The kinetic analysis reveals a small, slowly decaying, 2DG component that is not predicted by any of the models. If this component is treated as a separate, parallel compartment, then the four‐ and five‐parameter models are essentially equivalent. To compare our data to prior in vivo data, we combined 2DG and 2DG6P to produce Ci*, the total slice radioactivity, and analyzed the first 45 min of uptake. These data were fit best by a three‐parameter model and the slowly decaying pool was not identified. Calculation of glucose utilization from total tissue radioactivity, measured by whole slice homogenization and by image analysis of au‐toradiograms, showed excellent correlation between the two methods. Image analysis of radioactivity in the suprachias‐matic nucleus, which is present in these slices, revealed a spontaneous diurnal variation in in vitro glucose utilization in close quantitative agreement with prior in vivo measurements. The kinetic analysis of the 1,000 μm slice was qualitatively similar to that of the 540 μm slice but revealed an increase in the LC and a large decrease in k1 as well as the expected large increase in the hexokinase rate constant, k3. Overall, in vitro glucose utilization increased by about 60%. These results are consistent with our prior studies of the 1,000 μm slice and support our interpretation that the 1,000 μm slice is an excellent in vitro model for brain ischemia without infarction.

Diffusion of Radiotracers in Normal and Ischemic Brain Slices

Diffusion in the extracellular space (ECS) is important in physiologic and pathologic brain processes but remains poorly understood. To learn more about factors influencing tissue diffusion and the role of diffusion in solute-tissue interactions, particularly during cerebral ischemia, we have studied the kinetics of several radiotracers in control and hypoxic 450-μm hippocampal slices and in 1,050-μm thick slices that model the ischemic penumbra. Kinetics were analyzed by nonlinear least squares methods using models that combine extracellular diffusion with tissue compartments in series or in parallel. Studies with 14C-polyethylene glycol confirmed prior measurements of extracellular volume and that ECS shrinks during ischemia. Separating diffusion from transport also revealed large amounts of 45Ca that bind to or enter brain as well as demonstrating a small, irreversibly bound compartment during ischemia. The rapidity of 3H2O entry into cells made it impossible for us to distinguish intracellular from extracellular diffusion. The diffusion-compartment analysis of 3-O-methylglucose data appears to indicate that 5 mmol/L glucose is inadequate to support glycolysis fully in thick slices. Unexpectedly, the diffusion coefficient for all four tracers rose in thick slices compared with thin slices, suggesting that ECS becomes less tortuous in the penumbra.

Effects of calcium ions on glucose utilization in the rat suprachiasmatic nucleus in vitro

The role of calcium ions in maintenance of the circadian rhythm in glucose utilization in the suprachiasmatic nucleus (SCN) of the hypothalamus was investigated in vitro in a rat hypothalamic slice preparation using the 2-deoxyglucose (2-DG) method. In normal Krebs solution, 2-DG uptake of adult and embryonic day 22 rat SCN was higher in subjective day than in subjective night. In calcium-free Krebs solution, however, 2-DG uptake of adult and embryonic SCN was low in both subjective day and night periods. These results indicate that the SCN rhythm in metabolic activity is dependent on calcium ions in both adult and embryonic rats. Since immunohistochemical and ultrastructural analysis of synapse formation has shown very few synapses in the SCN of embryonic day 22 rats, it is suggested that the development and maintenance of the circadian rhythm in metabolism demonstrated by the 2-DG method depends on intracellular calcium-mediated events rather than synaptic transmission.

Kinetic Model of 2-Deoxyglucose Metabolism Using Brain Slices

A six-compartment, nine-parameter kinetic model of 2-deoxyglucose (2DG) metabolism, which includes bidirectional tissue transport, phosphorylation, two-step dephosphorylation, phosphoisomerization, and conjugation to UDP and macromolecules, has been derived. Data for analysis were obtained from 540- and 1,000-μm-thick hippocampal and hypothalamic brain slices, which were incubated in buffer containing [14C]2DG, frozen, extracted with perchlorate, and separated on anion-exchange columns. Solutions of the equations of the model were fit to the data by means of nonlinear least-squares analysis. These studies suggest that dephosphorylation is adequately described by a single reaction so that the model reduces to eight parameters. The in vitro rate constants for transport, phosphorylation, and dephosphorylation are very similar to prior in vivo results. The phosphoisomerization rate constant is similar to dephosphorylation, so glycosylated macromolecules slowly accumulate and gradually assume larger relative importance as other compounds disappear more rapidly. Rate constants for 540-μm slices from hypothalamus and hippocampus are similar, while 1,000-μm slices have smaller tissue transport constants and larger phosphorylation constants. The rate equation for glucose utilization of this model is relatively insensitive to uncertainties regarding the rate constants. Including later metabolic components in kinetic models improves the calculations of glucose utilization with long isotope exposures.

Restoring Adenine Nucleotides in a Brain Slice Model of Cerebral Reperfusion

Tissue adenine nucleotides are depleted during cerebral ischemia, impeding recovery after reperfusion. Although prior studies have attempted to prevent the initial loss of adenylates, the present study tests the hypothesis that stimulating synthesis of adenine nucleotides, through either adenosine kinase or adenine phosphoribosyltransferase, would result in significant cerebroprotection. To study the effects on neurons and glia directly while avoiding the influence of the cerebral vasculature, hippocampal brain slices were used for the model of transient ischemia with reperfusion. The standard brain slice insult of brief exposure to anoxia with aglycemia was modified based on studies which showed that a 30-minute exposure to air with 1 mmol/L glucose produced a stable, moderate reduction in ATP during the insult and that, 2 hours after return to normal conditions, there was moderate depletion of tissue adenine nucleotides and histologic injury. Treatments with 1 mmol/L adenosine, AMP, or adenine were equivalent in partially re-storing adenine nucleotides. Despite this, only adenosine af-forded histologic protection, suggesting a protective role for adenosine receptors. There also was evidence for metabolic cycling among adenine nucleotides, nucleosides, and purines. Adenosine may exert direct cerebroprotective effects on neural tissue as well as indirect effects through the cerebral vasculature.

Cerebral blood volume measurements by rapid contrast infusion and T 2*‐weighted echo planar MRI

Cerebral blood volume (CBV) provides information complementary to that of cerebral blood flow in cerebral ischemia, tumors, and other conditions. We have developed an alternative theory and method for measuring CBV based on dynamic imaging by MRI or CT during a short contrast infusion. This method avoids several limitations of traditional approaches that involve waiting for steady state or measuring the area under the curve (AUC) during bolus contrast injection. Anesthetized dogs were studied by T  2* ‐weighted echo planar imaging during gadolinium‐DTPA infusions lasting 30–60 sec. CBV was calculated from the ratio of the signal changes in tissue and artery. Method responsiveness was compared to AUC measurements using the vasodilator acepromazine. The ratio of signal change in tissue to that in artery rapidly approached an asymptotic value even while the amount of contrast in artery continued to increase. Using 30‐sec infusions, the mean (± SD) of CBV for control animals was 3.6 ± 0.9 ml blood/100 g tissue in gray matter and 2.3 ± 0.8 ml blood/100 g tissue in white matter (ratio = 1.6). Acepromazine increased CBV to 5.7 ± 1.5 ml blood/100 g tissue in gray matter and 3.1 ± 0.8 ml blood/100 g tissue in white matter (ratio = 2.0). AUC measurements after bolus injection yielded similar values for control animals but failed to demonstrate any change after acepromazine. It is possible to measure CBV using dynamic MRI or CT during 30–60‐sec contrast infusions. This method may be more sensitive to changes in CBV than traditional AUC methods. Magn Reson Med 47:1145–1157, 2002.

Time Course of ADCW Changes in Ischemic Stroke

To the Editor: Our laboratory team was excited to learn of the observation that there is an early increase in apparent diffusion coefficient (ADC) at the edge of ischemic lesions during stroke.1 We have recently completed a study that actually predicts this result and provides a potential explanation.2 Using 1000-μm-thick hippocampal brain slices as a model of the ischemic penumbra and a series of radiotracer molecules, we observed an increase in the rate of extracellular diffusion under ischemic conditions. Even though the extracellular space (ECS) was modestly reduced in volume in these slices, the diffusion coefficient rose with all …