Lactate, not pyruvate, is neuronal aerobic glycolysis end product: an in vitro electrophysiological study.

For over 60 years, a distinction has been made between aerobic and anaerobic glycolysis based on their respective end products: pyruvate of the former, lactate of the latter. Recently we hypothesized that, in the brain, both aerobic and anaerobic glycolysis terminate with the formation of lactate from pyruvate by the enzyme lactate dehydrogenase (LDH). If this hypothesis is correct, lactate must be the mitochondrial substrate for oxidative energy metabolism via its oxidation to pyruvate, plausibly by a mitochondrial LDH. Here we employed electrophysiology of the rat hippocampal slice preparation to test and monitor the effects of malonate and oxamate, two different LDH inhibitors, and glutamate, a neuronal activator, in experiments, the results of which support the hypothesis that lactate, at least in this in vitro setting, is indeed the principal end product of neuronal aerobic glycolysis.

Intermittent hypoxic exposure during light phase induces changes in cAMP response element binding protein activity in the rat CA1 hippocampal region: water maze performance correlates.

Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.

Preischemic hyperglycemia-aggravated damage: evidence that lactate utilization is beneficial and glucose-induced corticosterone release is detrimental.

Aerobic lactate utilization is crucial for recovery of neuronal function posthypoxia in vitro. In vivo models of cerebral ischemia pose a conceptual challenge when compared to in vitro models. First, the glucose paradox of cerebral ischemia, namely, the aggravation of delayed neuronal damage by preischemic hyperglycemia, cannot be reproduced in vitro. Second, in vitro elevated glucose levels protect against ischemic (hypoxic) damage, an outcome that has seldom been reproduced in vivo. Employing a rat model of cardiac-arrest-induced transient global cerebral ischemia (TGI), we found that hyperglycemic conditions, when induced 120-240 min pre-TGI, significantly reduced post-TGI neuronal damage as compared to normoglycemic conditions. In contrast, hyperglycemia, when induced 15-60 min pre-TGI, significantly aggravated post-TGI neuronal damage. Brain lactate levels in rats loaded with glucose either 15 min or 120 min pre-TGI were significantly and equally higher than those of control, saline-injected rats. The beneficial effect of 120 min pre-TGI glucose loading was abolished by lactate transport inhibition. A significant increase in blood corticosterone (CT) levels was observed upon glucose loading that peaked at 15-30 min and returned to baseline levels by 60-120 min. When rats loaded with glucose 15 min pre-TGI were treated with metyrapone, a CT synthesis inhibitor, a significantly lower degree of delayed neuronal damage in comparison to both untreated, 15 min glucose-loaded rats and normoglycemic, control rats was observed. Thus, although elevated levels of brain lactate cannot explain the glucose paradox of cerebral ischemia, hyperglycemia-induced, short-lived elevation in CT blood levels could. More importantly, lactate appears to play a crucial role in improving postischemic outcome.

Excitotoxic preconditioning elicited by both glutamate and hypoxia and abolished by lactate transport inhibition in rat hippocampal slices.

Ischemic preconditioning (PC) of heart and brain is a well-documented phenomenon. However, the mechanism underlying the increased resistance to severe ischemia by a preceding mild ischemic exposure remains unclear. Over a decade ago, we demonstrated the existence of hypoxic PC in the hippocampal slice preparation. Here we report the ability of a short exposure to toxic levels of glutamate to heighten the tolerance of hippocampal slices to a subsequent, longer exposure to the excitotoxin. Glutamate PC could also be induced by a short hypoxic exposure, suggesting a common mechanistic pathway for all PC stimuli. Since glutamate receptor activation and hypoxia increase tissue lactate production, a-cyano-4-hydroxycinnamate was applied during the PC period to completely abolished PC. These results indicate that excitotoxic PC and hypoxic PC share similar mechanisms that possibly involve lactate production and its neuronal utilization.

The mRNA level of the potassium-chloride cotransporter KCC2 covaries with seizure susceptibility in inferior colliculus of the post-ischemic audiogenic seizure-prone rat.

Cardiac arrest and resuscitation were used to induce brain damage and susceptibility to sound-triggered seizures in Sprague-Dawley rats. Glucose preloading was used to vary seizure susceptibility. Because loop diuretics can block these seizures, we investigated changes in KCC2, a potassium-chloride cotransporter, in the inferior colliculus - the origin of the seizures. Using polymerase chain reaction (PCR), we found that collicular KCC2 mRNA levels covaried with seizure susceptibility in these animals. Using quantitative PCR, we found that a fivefold increase in collicular KCC2 mRNA levels was associated with a doubling of seizure incidence. A hypothesis linking KCC2 activity to seizure susceptibility is presented.

Corticosterone-aggravated ischemic neuronal damage in vitro is relieved by vanadate.

Preischemic hyperglycemia-aggravated neuronal damage has been postulated to occur via enhanced lactic acidosis. We have hypothesized that preischemic glucose loading induces a short-lived elevation in glucocorticoid release which, when combined with ischemia, aggravates the postischemic outcome. This study tested this hypothesis in rat hippocampal slices exposed to 4 min in vitro ischemia of which 58% exhibited recovery of neuronal function. However, when corticosterone (CT) was present during ischemia, the recovery of neuronal function decreased in a concentration-dependent manner. At 5 microM, CT reduced the recovery rate to 40% while only 10% of slices recovered when exposed to 20 microM CT. Insulin could not block the effect of CT; however, vanadate improved the postischemic recovery of CT-treated (20 microM) slices to 43%. These results indicate that acute, short exposure to CT can significantly exacerbate postischemic outcome and that vanadate can antagonize CT action.

Blockade of lactate transport exacerbates delayed neuronal damage in a rat model of cerebral ischemia.

Studies over the past decade have demonstrated that lactate is produced aerobically during brain activation and it has been suggested to be an obligatory aerobic energy substrate postischemia. It has been also hypothesized, based on in vitro studies, that lactate, produced by glia in large amounts during activation and/or ischemia/hypoxia, is transported via specific glial and neuronal monocarboxylate transporters into neurons for aerobic utilization. To test the role of lactate as an aerobic energy substrate postischemia in vivo, we employed the cardiac-arrest-induced transient global cerebral ischemia (TGI) rat model and the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamate (4-CIN). Once 4-CIN was establish to cross the blood--brain barrier, rats were treated with the inhibitor 60 min prior to a 5-min TGI. These rats exhibited a significantly greater degree of delayed neuronal damage in the hippocampus than control, untreated rats, as measured 7 days post-TGI. We concluded that intra-ischemically-accumulated lactate is utilized aerobically as the main energy substrate immediately postischemia. Blockade of lactate transport into neurons prevents its utilization and, consequently, exacerbates delayed ischemic neuronal damage.

Ouabain induction of cycling of multiple spike responses in hippocampal slices is delayed by lithium.

Alterations in sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase) activity have been associated with changes of mood states and lithium treatment in bipolar illness. We examined the effects of ouabain and lithium on evoked population responses in rat hippocampal slices. In vitro 3.3 microM ouabain induced cycling between epliptiform activity and unresponsiveness in 18.5% of slices. In vitro ouabain, at 1-10 microM, induced epileptiform multiple spike responses. In vivo lithium pretreatment for 10-21 days produced a significant delay in the onset of this ouabain-induced epileptiform activity compared to control animals. These findings are consistent with other work which suggests that Na, K-ATPase inhibition can both activate and suppress excitable tissues and that lithium pretreatment can mitigate these effects. The implications of these results and others regarding the pathophysiology of bipolar illness are discussed.

Mechanical property predictions for polymorphs of sulphathiazole and carbamazepine.

The mechanical properties of polymorphs of sulphathiazole and carbamazepine have been determined experimentally by three-point beam bending. It is shown that the mechanical properties of sulphathiazole and carbamazepine polymorphs can be predicted using the atom-atom potential model applied to lattice dynamics, as long as account is taken of crystal morphology when considering the experimental results.

Study of cerebral energy metabolism using the rat hippocampal slice preparation.

This article describes methods and experimental paradigms used in combination with the rat hippocampal slice preparation in an attempt to better understand cerebral energy metabolism under the following conditions: normal resting conditions, conditions of oxygen and/or glucose deprivation, and conditions of activation (excitation). The outcome of this attempt, as described herewith, demonstrates the unmatched usefulness of the brain slice preparation as an in vitro tool in the field of neuroscience.

Examples of successful crystal structure prediction: polymorphs of primidone and progesterone.

The field of crystal structure prediction and its potential value to the pharmaceutical industry is described. The process of structure prediction employed here is summarized and the results of its application to primidone and progesterone are reported. It is shown that the process successfully generates the known polymorphs of these molecules, starting from the molecular structure alone. Observations related to the application of the structure prediction process are reported.

Correlates of delayed neuronal damage and neuroprotection in a rat model of cardiac-arrest-induced cerebral ischemia.

Numerous studies over the past three decades have used rodent models of cerebral ischemia. To measure the postischemic outcome, the majority of these studies used histopathology as the method of choice both quantitatively and qualitatively. No functional measure of postischemic outcome has been proved to correlate well with the histopathological one. The rat chest compression model of cardiac-arrest-induced global cerebral ischemia was used in the present study. Two separate measures of neuronal damage at 7 days postischemia were performed: (a) histologically, by counting normal pyramidal cell bodies in the mid-CA1 hippocampal region of the rat brain, in hematoxylin-eosin-stained, paraffin-embedded 6-microm sections, and (b) electrophysiologically, by counting the number of 400 microm hippocampal slices in which it was possible to evoke a normal (>/=10 mV) CA1 population spike by orthodromic stimulation of the Schaffer collaterals. The correlation between these two measures was tested in the following groups of rats: (a) control, untreated group, (b) MK-801-treated groups (0.03 to 1.0 mg/kg given i.p. shortly after ischemia), (c) diltiazem-treated (DILT) groups 1.0 to 30 mg/kg, given i.p. shortly after ischemia, and (d) a group treated with a combination of the two drugs together (0.1 mg/kg MK-801+3.0 mg/kg DILT given i.p. shortly after ischemia). The two measures of postischemic outcome were highly correlated in all groups studied. Both MK-801 and DILT exhibited a dose-dependent neuroprotective effect. When administered together, a synergy between the neuroprotective effect of MK-801 and DILT was observed. At the doses used, minimal or no side effects of either MK-801 or DILT were observed.

An increase in lactate output by brain tissue serves to meet the energy needs of glutamate-activated neurons.

Aerobic energy metabolism uses glucose and oxygen to produce all the energy needs of the brain. Several studies published over the last 13 years challenged the assumption that the activated brain increases its oxidative glucose metabolism to meet the increased energy demands. Neuronal function in rat hippocampal slices supplied with 4 mM glucose could tolerate a 15 min activation by a 5 mM concentration of the excitatory neurotransmitter glutamate (Glu), whereas slices supplied with 10 mM glucose could tolerate a 15 min activation by 20 mM Glu. However, in slices in which neuronal lactate use was inhibited by the lactate transporter inhibitor a-cyano-4-hydroxycinnamate (4-CIN), activation by Glu elicited a permanent loss of neuronal function, with a twofold to threefold increase in tissue lactate content. Inhibition of glycolysis with the glucose analog 2-deoxy-D-glucose (2DG) during the period of exposure to Glu diminished normal neuronal function in the majority of slices and significantly reduced the number of slices that exhibited neuronal function after activation. However, when lactate was added with 2DG, the majority of the slices were neuronally functional after activation by Glu. NMDA, a nontransportable Glu analog by the glial glutamate transporter, could not induce a significant increase in slice lactate level when administered in the presence of 4-CIN. It is suggested that the heightened energy demands of activated neurons are met through increased glial glycolytic flux. The lactate thus formed is a crucial aerobic energy substrate that enables neurons to endure activation.

The glucose paradox in cerebral ischemia. New insights.

The present in vivo findings that lactate, accumulated during an ischemic episode, is an essential aerobic energy substrate during the initial postischemic period are in full agreement with out in vitro findings. Moreover, the beneficial effects of hyperglycemia are also in agreement with our and others' in vitro results that have demonstrated a neuroprotective effect of glucose against hypoxic change. The aggravation of ischemic delayed neuronal damage by glucose loading 15 min prior to the ischemic insult is likely the result of glucose induction of a short-acting (30 to 60 min) systemic factor (hormonal?) that, when combined with an ischemic insult, potentiates the ischemic damage.

Brain lactate is an obligatory aerobic energy substrate for functional recovery after hypoxia: further in vitro validation.

This study used the rat hippocampal slice preparation and the monocarboxylate transporter inhibitor, alpha-cyano-4-hydroxycinnamate (4-CIN), to assess the obligatory role that lactate plays in fueling the recovery of synaptic function after hypoxia upon reoxygenation. At a concentration of 500 microM, 4-CIN blocked lactate-supported synaptic function in hippocampal slices under normoxic conditions in 15 min. The inhibitor had no effect on glucose-supported synaptic function. Of control hippocampal slices exposed to 10-min hypoxia, 77.8 +/- 6.8% recovered synaptic function after 30-min reoxygenation. Of slices supplemented with 500 microM 4-CIN, only 15 +/- 10.9% recovered synaptic function despite the large amount of lactate formed during the hypoxic period and the abundance of glucose present before, during, and after hypoxia. These results indicate that 4-CIN, when present during hypoxia and reoxygenation, blocks lactate transport from astrocytes, where the bulk of anaerobic lactate is formed, to neurons, where lactate is being utilized aerobically to support recovery of function after hypoxia. These results unequivocally validate that brain lactate is an obligatory aerobic energy substrate for posthypoxia recovery of function.

Brain lactate, not glucose, fuels the recovery of synaptic function from hypoxia upon reoxygenation: an in vitro study.

Lactate has been considered for many years to be a useless, and frequently, harmful end-product of anaerobic glycolysis. In the present in vitro study, lactate-supplied rat hippocampal slices showed a significantly higher degree of recovery of synaptic function after a short hypoxic period than slices supplied with an equicaloric amount of glucose. More importantly, all slices in which anaerobic lactate production was enhanced by pre-hypoxia glucose overload exhibited functional recovery after a prolonged hypoxia. An 80% recovery of synaptic function was observed even when glucose utilization was blocked with 2-deoxy-D-glucose during the later part of the hypoxic period and during reoxygenation. In contrast, slices in which anaerobic lactate production was blocked during the initial stages of hypoxia did not recover their synaptic function upon reoxygenation despite the abundance of glucose and the removal of 2-deoxy-D-glucose. Thus, for brain tissue to show functional recovery after prolonged period of hypoxia, the aerobic utilization of lactate as an energy substrate is mandatory.

Glia are the main source of lactate utilized by neurons for recovery of function posthypoxia.

Experiments are described in which a rat hippocampal slice preparation was used along with the metabolic glial inhibitor, fluorocitrate (FC), to investigate the role of glial-made lactate and its shuttling to neurons in posthypoxia recovery of synaptic function. After testing two less effective concentrations of FC, only 10.1 +/- 6.5% of slices treated with 100 microM of the metabolic toxin recovered synaptic function at the end of 10-min hypoxia and 30-min reoxygenation. In contrast, 79.6 +/- 7.4% of control, untreated slices recovered synaptic function after 10-min hypoxia and 30-min reoxygenation. The low rate of recovery of synaptic function posthypoxia in FC-treated slices occurred despite the abundance of glucose present in the medium before, during, and after hypoxia. The amount of lactate produced by FC-treated slices during the hypoxic period was only 62% of that produced by control, untreated slices. Supplementing FC-treated slices with exogenous lactate significantly increased the posthypoxia recovery rate of synaptic function. These results strongly support our previous findings concerning the mandatory role of lactate as an aerobic energy substrate for the recovery of synaptic function posthypoxia and clearly show that the bulk of the lactate needed for this recovery originates in glial cells.

Cell swelling exacerbates hypoxic neuronal damage in rat hippocampal slices.

In the present study we investigated the effect of acute cell swelling on the sensitivity of rat hippocampal slices to hypoxia. Hippocampal slices were exposed to different degrees of hypo- or hyperosmolality 15 min prior to and during a 15-min hypoxia followed by reoxygenation under isosmotic (293 mOsm) conditions. Recovery of neuronal function (an electrically evoked population spike) after hypoxia was significantly diminished in slices exposed to hyposmotic conditions as 57% of control (isosmotic) slices showed recovery compared with 51%, 35%, and 13% recovery rate in slices made hyposmotic (273, 253, and 233 mOsm, respectively). Of slices exposed to a medium made hyperosmotic by the addition of 20, 40, 60, and 80 mM mannitol, only those exposed to the most hyperosmotic treatment (373 mOsm) exhibited a recovery rate significantly greater than control (70% vs. 57%). The competitive NMDA antagonist CGS-19755 (50 microM) completely protected both isosmotic and hyposmotic (233 mOsm) slices against hypoxic damage. However, a threshold dose (15 microM) of the antagonist provided no protection to isosmotic slices (51% vs. 57% recovery rate) while affording substantial protection to hyposmotic slices (233 mOsm), as 54% of the treated slices recovered their neuronal function after hypoxia compared to 13% recovery rate of the untreated slices. These results suggest an increase in activation of the NMDA receptor under hyposmotic conditions. We conclude that acute osmotic swelling of neuronal tissue predisposes it to hypoxic damage, possibly by activation of NMDA receptors that are not usually activated by hypoxia alone.

Freeze-drying: in situ observations using cryoenvironmental scanning electron microscopy and differential scanning calorimetry.

The quality of a freeze-dried product and the ease with which it can be reconstituted depend upon the morphology which develops during freezing and upon solvent sublimation. In this paper, the use of an environmental scanning electron microscope (ESEM) with a cryostage attachment is reported as a means of studying such structural evolution during the sublimation process. Results indicate that cryo-ESEM can provide a useful addition to techniques already used for the development of freeze-drying cycles, such as differential scanning calorimetry and light microscopy.

Audiogenic seizures following global ischemia induced by chest compression in Long-Evans rats.

Transient global ischemia was used to produce a rat model of generalized tonic-clonic epilepsy. Controlled chest compression in ketamine-anesthesized Long-Evans rats produced transient global ischemia by mechanically preventing the heart from pumping blood. Circulation was restored by standard cardiopulmonary resuscitation techniques. With a temporal muscle (skull) temperature of 35 +/- 0.4 degrees C, 75% (76/102) of the rats survived 7 min of chest compression. Generalized seizures could be evoked in 78% (59/76) of the surviving rats by a 60 s exposure to a loud sound (bell, 110 dB) beginning 24 h after the ischemic episode. The seizure patterns seen resembled those described by Maresceaux (1987) for genetically seizure-prone Wistar rats. Susceptibility to sound-induced seizures declined with time, with wide variations in recovery rate between individuals; one rat showed a daily sound-induced seizure for over 5 months. Seizures were attenuated or blocked by treatment with carbamazepine or sodium valproate. This model is similar to the great vessel occlusion model used by Kawai et al. (1995), but is less invasive. We believe it will be useful in the evaluation of therapies for acquired generalized (grand mal) seizures.