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.

Pelvic cancer pain.

Pelvic cancer causes several types of pain, i.e., visceral, neuropathic, and somatic pain. Somatic pain is due to stimulation of nociceptors in the integument and supporting structures, namely, striated muscles, joints, periosteum, bones, and nerve trunks by direct extension through fascial planes and their lymphatic supply. In 60% of patients with malignant disease of soft tissues, nerve trunk, and sacral invasion from carcinoma of the cervix, uterus, vagina, colon, rectum, and other tissues in women, and from penile, prostate, and colorectal carcinoma and sarcoma in men, they have neuropathic pain. The infiltration of the perineal nerves results in lumbosacral plexopathies and complete destruction of the nerve, including perineural lymphatic invasions producing symptomatic sensory loss, causalgia, and deafferentation. Visceral pain is the result of spasms of smooth muscles of hallow viscus; distortion of capsule of solid organs; inflammation; chemical irritation; traction or twisting of mesentery; and ischemia, or necrosis, and encroachment of pelvis and presacral tumors. Pain of these types is managed by different modalities depending on the age of the patient, the expected life expectancy, availability of invasive and non-invasive pain control modalities, and the resources of the patient, community, and health care agencies. Patients with pelvic cancer can live with less pain due to better pain-control modalities that are available today with the help of dedicated and caring algologists.

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.

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 anaerobic lactate production: a suicide note or a survival kit?

Aerobic energy metabolism utilizes glucose and oxygen to satisfy all the energy needs of the adult brain. Anaerobically, the brain switches to the significantly less efficient glycolytic pathway for its most basic energy requirements. Anaerobic glycolysis provides the adult brain with a limited amount of energy and time to maintain ion homoeostasis and other essential processes before several events occur that lead to brain cell damage and death. Recent evidence that lactate, produced mainly in glial cells during a period of oxygen deprivation, becomes the only utilizable and thus obligatory substrate for aerobic energy metabolism upon reoxygenation is summarized here. This evidence also supports the hypothesis that a lactate shuttle exists between glia and neurons, and emphasizes its importance in the post-ischemic survival of neurons.

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.

Cardiac arrest-induced global cerebral ischemia studied in vitro.

The goal of the present study was to characterize the effects of chest compression-induced global cerebral ischemia on the hippocampal slice preparation. One of the characteristics of rats exposed to such cardiac arrest is a high susceptibility to sound-induced seizures. We tested audiogenic seizures as an in vivo indicator of ischemic cerebral damage and as a possible small animal model of epilepsy. The results of these tests were reported elsewhere. Long-Evans male rats (200-350 g) were subjected to 7 min of chest compression sufficient to stop the pumping action of the heart. The rats were then revived using cardiopulmonary resuscitation. Evaluation of cerebral damage following cardiac arrest and resuscitation was performed in vitro, by testing neuronal responses to electrical stimulation in hippocampal slices prepared from these animals. Sham control animals were used for comparisons. Twenty-one to 146 days after rats were chest-compressed, hippocampal slices were prepared. Sham control rats, anesthetized but not chest-compressed, were sacrificed one week later for preparation of slices. Rats in a second group exposed to 7-min chest compression, were sacrificed at different time intervals after their resuscitation (from 1 h to 7 days); hippocampal slices were prepared for electrophysiological analysis of neuronal damage. The results of these studies indicate that 3 weeks or longer after chest compression the evoked CA1 population spike amplitude in hippocampal slices was significantly attenuated; in 60% of these slices an epileptiform response was evoked. An increased proportion of slices prepared from rats 1 to 48 h after chest compression showed an augmentation in the amplitude of the evoked population spike; 72 h and up to 7 days after chest compression, an attenuation in the evoked CA1 population spike amplitude was observed, signaling delayed neuronal damage.

Synergism between diltiazem and MK-801 but not APV in protecting hippocampal slices against hypoxic damage.

In the present study, we investigated the possibility that MK-801 (dizocilpine), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, owes its potent neuroprotective properties to calcium channel blocking ability rather than to its NMDA receptor antagonism. Rat hippocampal slices were exposed to a long hypoxic period (20 min) from which only 13.8% recovered their neuronal function after 30 min of reoxygenation. The recovery rate of neuronal function from 20-min hypoxia was increased to 100% when slices were pretreated with 5 microM MK-801. DL-2-amino-5-phosphonovalerate (APV), a competitive NMDA receptor antagonist, even at relatively high concentration (100 microM), provided only marginal protection against such severe hypoxic insult. The L-type calcium channel blocker diltiazem (DILT) was more effective than APV in protecting hypoxic slices against neuronal damage. Combining suboptimal concentrations of DILT and MK-801 produced a neuroprotective effect with significantly exceeded the calculated additive effect of the two drugs. Such synergism could not be demonstrated between DILT and APV, a combination that produced only the expected additive neuroprotective effect. The observed synergy between the calcium channel blocker (DILT) and MK-801, along with other studies that demonstrated interaction between these two drugs, led us to postulate that MK-801 possesses calcium channel blocking properties through which its neuroprotective effect is exerted. These calcium channels could either be of the L-type or otherwise, channels which are being activated only under stressful conditions, such as hypoxia or ischemia.

Hypoxia, excitotoxicity, and neuroprotection in the hippocampal slice preparation.

The excitotoxic hypothesis postulates a central role for the excitatory amino acids (EAAs) and their receptors in the neuronal damage that ensues cerebral ischemia-hypoxia and numerous other brain disorders. A major premise of the excitotoxic hypothesis is that neuronal protection can be achieved via blockade of EAA receptors with specific antagonists. This paper describes the use of the rat hippocampal slice preparation in the evaluation of various EAAs and their analogues for their potency as excitotoxins (agonists) and antagonists of the NMDA and the kainate/AMPA glutamate receptor subtypes. The hypersensitivity of hypoxic hippocampal slices to the presence of excitotoxins provided us with an inexpensive, sensitive tool to distinguish between structurally similar compounds. Moreover, these studies indicate that hypoxic neuronal damage cannot solely result from an excitotoxic mechanism; the involvement of voltage-dependent calcium channels in such damage is likely, as is evident from experiments performed in calcium-depleted medium and with the non-competitive NMDA antagonist MK-801. At sub-toxic doses, quinolinate, a tryptophan metabolite implicated in Huntington's disease, appears to be a strong potentiator of the toxicity of all excitotoxins tested.

Protection by MK-801 against hypoxia-, excitotoxin-, and depolarization-induced neuronal damage in vitro.

Exposure of rat hippocampal slices to 12-min hypoxia produced only mild neuronal damage, as 72% of all slices recovered their CA1-evoked population spike following a 30-min recovery period. However, when this hypoxic insult was administered in the presence of 2.5 microM kainate or AMPA, only 6 and 15% of the slices, respectively, recovered their neuronal function. This enhancement of hypoxic damage by kainate could be attenuated in a dose-dependent fashion by the kainate/AMPA antagonist GYKI 52466 but not by the competitive NMDA antagonist APV. Unexpectedly, the noncompetitive NMDA antagonist MK-801 also attenuated the kainate- and AMPA-enhanced hypoxic neuronal damage and was more efficacious than GYKI 52466. Considering (1) the ability of MK-801 to antagonize hypoxic neuronal damage in the absence or the presence of NMDA, kainate or AMPA; (2) the antihypoxic effect of MK-801 in the presence of APV + 7-chlorokynurenate, a pairing that supposedly blocks MK-801 binding to the NMDA receptor; (3) the ability of MK-801 to protect hippocampal slices against brain damage induced by depolarization + excitotoxin (50 mM KCl + mM glutamate for 60 min); and (4) the ability of diltiazem, an L-type calcium channel blocker, to protect hippocampal slices against hypoxic neuronal damage, we conclude that the mode of action of MK-801 cannot be explained by its NMDA receptor antagonistic properties alone. A possible blockade of Ca2+ channels, most likely of the L-type, by MK-801 should be considered along with other mechanisms.

Sensory evoked facial muscle electromyography for the quantification of acute labor pain.

Assessment of the adequacy of epidural analgesia for acute pain management can be difficult on occasion. This investigation used non-invasive sensory evoked facial muscle electromyography (SEFE) as well as a Verbal Assessment Scores (VAS) to assess severe pain in healthy parturients during the first stage of active labor. Institutional Review Board approval and patient informed consent were obtained from 12 healthy parturients who were in active labor and who had requested epidural analgesia for labor pain. SEFE microvoltage was recorded prior to epidural placement when a patient reported severe pain and again when a patient reported no pain with a subsequent uterine contraction. VAS assessments (0 = no pain and 10 = the worst pain ever experienced) were also recorded at identical time intervals. Statistical analysis was done using the paired two tailed Student's t-test. Each patient served as their own control. A statistically significant decrease in SEFE microvoltage (p < 0.001) was noted when analgesia was established (VAS = 0) in each patient. It was concluded in this pilot study that SEFE can be effective in quantifying acute severe nociception and thus can provide a continuous objective indicator of the effectiveness of analgesic regimens in an acute obstetric pain setting. Its applicability in other acute pain areas remains to be investigated.

Quinolinate potentiates the neurotoxicity of excitatory amino acids in hypoxic neuronal tissue in vitro.

Excitatory amino acids (EAAs) in the central nervous system are involved in both neurotransmission and neurotoxicity. Quinolinate (QUIN) is a neurotoxic endogenous tryptophan metabolite that has been linked to Huntington's disease, Alzheimer's disease, and many inflammatory diseases. We used the rat hippocampal slice preparation and its electrophysiology to study the interaction of QUIN with glutamate receptor agonists such as N-methyl-D-aspartate (NMDA), glutamate, aspartate, kainate, and AMPA ((R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate). The majority of slices could tolerate an exposure to 10-min hypoxia (86% recovered their neuronal function), but doses of glutamate receptor agonists which were harmless under normoxic conditions, significantly reduced this recovery rate under hypoxic conditions. QUIN, at doses that even under hypoxic conditions were innocuous (20-50 microM), potentiated the neurotoxic effects of all the glutamate receptor agonists tested in hypoxic hippocampal slices. The NMDA antagonist D,L-2-amino-5-phosphonovalerate blocked this potentiation while 7-chlorokynurenate, at a dose sufficient to block the effect of NMDA alone, was ineffective in blocking the potentiation of NMDA toxicity by QUIN. Non-toxic analogues of QUIN (6-methyl-QUIN and 2,3-pyrazine dicarboxylate) were also able to potentiate NMDA toxicity in hypoxic slices. The results of these experiments provided indirect evidence that QUIN is an endogenous potentiator of the NMDA and the kainate receptor subtypes; therefore, we postulate that QUIN has a specific modulatory binding site on all glutamate receptor subtype complexes. Regardless of its site of interaction, the importance of QUIN as a potentiator of the agonistic activation of these receptors cannot be overemphasized.(ABSTRACT TRUNCATED AT 250 WORDS)

Kainate toxicity in energy-compromised rat hippocampal slices: differences between oxygen and glucose deprivation.

The effects of kainate (KA) on the recovery of neuronal function in rat hippocampal slices after hypoxia or glucose deprivation (GD) were investigated and compared to those of (R,S)-alpha-amino-3-hydroxy-5-methyl-4- isoxazoleproprionate (AMPA). KA and AMPA were found to be more toxic than either N-methyl-D-aspartate (NMDA), quinolinate, or glutamate, both under normal conditions and under states of energy deprivation. Doses as low as 1 microM KA or AMPA were sufficient to significantly reduce the recovery rate of neuronal function in slices after a standardized period of hypoxia or GD. The enhancement of hypoxic neuronal damage by both agonists could be partially blocked by the antagonist kynurenate, by the NMDA competitive antagonist AP5, and by elevating [Mg2+] in or by omitting Ca2+ from the perfusion medium. The AMPA antagonist glutamic acid diethyl ester was ineffective in preventing the enhanced hypoxic neuronal damage by either KA or AMPA. The antagonist of the glycine modulatory site on the NMDA receptor, 7-chlorokynurenate, did not block the KA toxicity but was able to block the toxicity of AMPA. 2,3-Dihydroxyquinoxaline completely blocked the KA- and AMPA-enhanced hypoxic neuronal damage. The KA-enhanced, GD-induced neuronal damage was prevented by Ca2+ depletion and partially antagonized by kynurenate but not by AP5 or elevated [Mg2+]. The results of the present study indicate that the KA receptor is involved in the mechanism of neuronal damage induced by hypoxia and GD, probably allowing Ca2+ influx and subsequent intracellular Ca2+ overload.(ABSTRACT TRUNCATED AT 250 WORDS)