NMDA-induced apoptosis in mixed neuronal/glial cortical cell cultures: the effects of isoflurane and dizocilpine

In animal models of severe ischemia, it has not been uniformly observed that anesthetics are protective. However, anesthetics have not been evaluated in the presence of a mild excitotoxic insult. We hypothesized that in the presence of a mild excitotoxic insult, 3 microm N-methyl-D-aspartate (NMDA), isoflurane may prevent apoptotic cell death. Primary mixed neuronal/glial cultures were prepared from fetal rat brains. Mature cultures were exposed to dissolved isoflurane [0 mM, 0.4 mM (1.8 minimum alveolar concentration) or 1.6 mM (7 minimum alveolar concentration)] or dizocilpine (10 microM), and NMDA (0 or 3 microM) at 37 degrees C for 30 minutes. Apoptosis was assessed using terminal-deoxy-nucleotidyl end-nick labeling oligonucleosomal DNA fragmentation enzyme-linked immunosorbent assay, and caspases-3 and -9 activation assays. NMDA (3 muM) induced apoptosis in mixed neuronal/glial cell cultures. Apoptosis induced by 3 microm NMDA was caspase-3 but not caspase-9 mediated. In the presence of a mild excitotoxic insult, this investigation showed an attenuation of apoptotic cell death by dizocilpine, but not isoflurane.     

Effects of volatile anesthetics on N-methyl-D-aspartate excitotoxicity in primary rat neuronal-glial cultures

BACKGROUND: Volatile anesthetics are known to ameliorate experimental ischemic brain injury. A possible mechanism is inhibition of excitotoxic cascades induced by excessive glutamatergic stimulation. This study examined interactions between volatile anesthetics and excitotoxic stress. METHODS: Primary cortical neuronal-glial cultures were exposed to N-methyl-D-aspartate (NMDA) or glutamate and isoflurane (0.1-3.3 mM), sevoflurane (0.1-2.9 mM), halothane (0.1-2.9 mM), or 10 microM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801). Lactate dehydrogenase release was measured 24 h later. In other cultures, effects of volatile anesthetics on Ca++ uptake and mitochondrial membrane potential were determined in the presence or absence of NMDA (0-200 microM). RESULTS: Volatile anesthetics reduced excitotoxin induced lactate dehydrogenase release by up to 52% in a dose-dependent manner. At higher concentrations, this protection was reversed. When corrected for olive oil solubility, the three anesthetics offered equivalent protection. MK-801 provided near-complete protection. Ca++ uptake was proportionally reduced with increasing concentrations of anesthetic but did not account for reversal of protection at higher anesthetic concentrations. Given equivalent NMDA-induced Ca++ loads, cells treated with volatile anesthetic had greater lactate dehydrogenase release than those left untreated. At protective concentrations, volatile anesthetics partially inhibited NMDA-induced mitochondrial membrane depolarization. At higher concentrations, volatile anesthetics alone were sufficient to induce mitochondrial depolarization. CONCLUSIONS: Volatile anesthetics offer similar protection against excitotoxicity, but this protection is substantially less than that provided by selective NMDA receptor antagonism. Peak effects of NMDA receptor antagonism were observed at volatile anesthetic concentrations substantially greater than those used clinically.     

Effect of isoflurane on neuronal apoptosis in rats subjected to focal cerebral ischemia

Although isoflurane can reduce ischemic neuronal injury after short postischemic recovery intervals, this neuroprotective efficacy is not sustained. Neuronal apoptosis can contribute to the gradual increase in infarct size after ischemia. This suggests that isoflurane, although capable of reducing early neuronal death, may not inhibit ischemia-induced apoptosis. We investigated the effects of isoflurane on markers of apoptosis in rats subjected to focal ischemia. Fasted Wistar-Kyoto rats were anesthetized with isoflurane and randomly allocated to awake (n = 40) or isoflurane (n = 40) groups. Animals in both groups were subjected to focal ischemia by filament occlusion of the middle cerebral artery for 70 min. Pericranial temperature was servo-controlled at 37 degrees C +/- 0.2 degrees C throughout the experiment. In the awake group, isoflurane was discontinued and the animals were allowed to awaken. In the isoflurane group, isoflurane anesthesia was maintained at 1.5 MAC (minimum alveolar anesthetic concentration). Animals were killed 7 h, 1 day, 4 days, or 7 days after reperfusion (n = 10/group/time point). The area of cerebral infarction was measured by image analysis in a hematoxylin and eosin stained section. In three adjacent sections, apoptotic neurons were identified by TUNEL staining and immunostaining for active caspase-9 and caspase-3. Infarct size was smaller in the isoflurane group than the awake group 7 h, 1 day, and 4 days after reperfusion (P < 0.05). However, this difference was absent 7 days after reperfusion. The number of apoptotic (TUNEL, caspase-3, and caspase-9 positive) cells 1 day after ischemia was significantly more in the awake versus isoflurane group. After a recovery period of 4 or 7 days, the number of apoptotic cells in the isoflurane group was more than in the awake group. After 7 days, the number of caspase-3 and -9 positive neurons was more in the isoflurane group (P < 0.05). The data indicate that isoflurane delays but does not prevent the development of cerebral infarction caused by ischemia. Isoflurane reduced the development of apoptosis early after ischemia but did not prevent it at later stages of postischemic recovery. IMPLICATIONS: The effect of isoflurane on neuronal apoptosis was investigated in rats subjected to focal cerebral ischemia. In isoflurane-anesthetized animals, ischemia-induced apoptosis occurred during the later stages of postischemic recovery. Isoflurane did not inhibit postischemic neuronal apoptosis.     

Isoflurane reduces N-methyl-D-aspartate toxicity in vivo in the rat cerebral cortex

Recent in vitro data indicate that isoflurane can reduce N-methyl-D-aspartate (NMDA) receptor-mediated responses and thereby might reduce excitotoxicity. However, the effect of isoflurane on NMDA receptor-mediated toxicity in vivo is not known. We conducted the present study to evaluate the effect of isoflurane on injury produced by cortical injection of NMDA in vivo and to compare it with dizocilpine, an antagonist of the NMDA receptor. Fasted Wistar-Kyoto rats were anesthetized with isoflurane. NMDA 50 nmoles (5-microL volume) were stereotactically injected into the cortex (2.8 mm lateral and 2.8 mm rostral to the bregma, depth 2 mm) of animals in one of four groups. In the isoflurane groups, the end-tidal concentration of isoflurane was maintained at either electroencephalogram (EEG)-burst suppression (BS) doses (2.2%-2.3%, n = 12) or a 1 minimum alveolar anesthetic concentration (MAC) dose (n = 10). In the dizocilpine group (n = 10), 10 mg/kg dizocilpine was injected IV 15 min before the NMDA injection. In the awake group and the dizocilpine group, anesthesia was discontinued on completion of the NMDA injection, and the animals were allowed to awaken. In the animals in the control group (n = 10), 20 microL of artificial cerebrospinal fluid was injected into the cortex. Injury to the cortex was evaluated 2 days after the NMDA injection. In 1 MAC doses and EEG-BS doses, isoflurane reduced the injury produced by a cortical NMDA injection compared with the awake state (1.74+/-0.49 and 0.96+/-0.46 vs 2.34+/-0.56 mm3; P = 0.02). Dizocilpine reduced cortical injury (0.56+/-0.27; P = 0.01) compared with the awake state. Injury in the control group was limited to the trauma produced by cannula insertion. In the isoflurane EEG-BS and dizocilpine groups, the injury was not different from the control group. IMPLICATIONS: Isoflurane can reduce N-methyl-D-aspartate-mediated cortical injury in vivo in a dose-dependent manner. These data are consistent with the previously demonstrated ability of isoflurane to reduce N-methyl-D-aspartate receptor-mediated responses in vitro.     

Volatile Anesthetics Selectively Inhibit the Calcium sup 2+ -transporting ATPase in Neuronal and Erythrocyte Plasma Membranes

Background: The activity of the plasma membrane Calcium2+ - transporting adenosine triphosphatase (PMCA) is inhibited by volatile anesthetics at clinical concentrations. The goal of the current study was to determine whether the inhibition is selective as compared to other adenosine triphosphatases (ATPases) and another group of general anesthetics, barbiturates. In addition, the authors determined whether the response to anesthetics of the enzymes in neuronal membranes is similar to that in erythrocyte membranes.

Methods: The effects of halothane, isoflurane, and sodium pentobarbital on four different ATPase activities were studied at 37 degrees C in two distinct plasma membrane preparations, human red blood cells and synaptosomal membranes from rat cerebellum.

Results: Inhibition patterns of the PMCA by halothane and isoflurane at anesthetic concentrations were very similar in red blood cells and synaptosomal membranes. The half-maximal inhibition (I50) occurred at 0.25-0.30 mM halothane and 0.30-0.32 mM isoflurane. The PMCA in both membranes was significantly more sensitive to the inhibitory action of volatile anesthetics (I50 = 0.75-1.15 minimum alveolar concentration) than were other ATPases, such as the Sodium sup +, Potassium sup + -ATPase (I50 [nearly equal] 3 minimum alveolar concentration) or Magnesium sup 2+ -ATPase (I50 greater or equal to 5 minimum alveolar concentration). In contrast, sodium pentobarbital inhibited the PMCA in both membranes only at [nearly equal] 100-200-fold above its anesthetic concentrations. The other ATPases were inhibited at similar pentobarbital concentrations (I50 = 11-22 mM).     

Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain

BACKGROUND: Anesthetics, including isoflurane and nitrous oxide, an antagonist of the N-methyl-D-aspartate subtype of the glutamate receptor, have been demonstrated to induce apoptotic neurodegeneration when administered during neurodevelopment. Xenon, also an N-methyl-D-aspartate antagonist, not only lacks the characteristic toxicity produced by other N-methyl-D-aspartate antagonists, but also attenuates the neurotoxicity produced by this class of agent. Therefore, the current study sought to investigate xenon's putative protective properties against anesthetic-induced neuronal apoptosis. METHOD: Separate cohorts (n = 5 or 6 per group) of 7-day-old rats were randomly assigned and exposed to eight gas mixtures: air, 75% nitrous oxide, 75% xenon, 0.75% isoflurane, 0.75% isoflurane plus 35% or 75% nitrous oxide, 0.75% isoflurane plus 30% or 60% xenon for 6 h. Rats were killed, and cortical and hippocampal apoptosis was assessed using caspase-3 immunostaining. In separate cohorts, cortices were isolated for immunoblotting of caspase 3, caspase 8, caspase 9, and cytochrome c. Organotypic hippocampal slices of postnatal mice pups were derived and cultured for 24 h before similar gas exposures, as above, and subsequently processed for caspase-3 immunostaining. RESULTS: In vivo administration of isoflurane enhances neuronal apoptosis. When combined with isoflurane, nitrous oxide significantly increases whereas xenon significantly reduces apoptosis to a value no different from that of controls. In vitro studies corroborate the ability of xenon to attenuate isoflurane-induced apoptosis. Isoflurane enhanced expression of indicators of the intrinsic and common apoptotic pathways; this enhancement was increased by nitrous oxide but attenuated by xenon. CONCLUSIONS: The current study demonstrates that xenon prevents isoflurane-induced neonatal neuronal apoptosis.     

Norepinephrine-induced apoptosis is inhibited in adult rat ventricular myocytes exposed to volatile anesthetics

BACKGROUND: Volatile anesthetics are used to provide anesthesia to patients with heart disease under heightened adrenergic drive. The purpose of this study was to test whether volatile anesthetics can inhibit norepinephrine (NE)-induced apoptosis in cardiomyocytes. METHODS: Rat ventricular cardiomyocytes were exposed to NE (10 microm) alone or in the presence of increasing concentrations of isoflurane and halothane. RESULTS: Isoflurane at 1.6 minimum alveolar concentration (MAC) (4 +/- 2% [SD]) and halothane at 1.2 MAC (3 +/- 2%) abolished the percentage of cardiomyocytes undergoing NE-induced apoptosis (34 +/- 8%), as assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) (P < 0.0001). Lower concentrations of isoflurane and halothane markedly decreased the number of TUNEL-positive cells. Similarly, isoflurane at 1.6 MAC (5 +/- 3%) and halothane at 1.2 MAC (6 +/- 3%) prevented the increase in annexinV-staining cardiomyocytes (38 +/- 7%; P < 0. 0001). These findings were corroborated with a decreased quantity of NE-induced DNA laddering by volatile anesthetics. Halothane at 1.2 MAC abolished the increase in TUNEL-positive cardiomyocytes exposed to the dihydropyridine Ca2+-channel agonist BAY K-8644 (1 microm) (BAY K-8644 + halothane: 3 +/- 2% vsBAY K-8644: 34 +/- 6%; P < 0. 0001) and the Ca2+-ionophore 4-bromo-A23187 (1 microm) (4-bromo-A23187 + halothane: 2 +/- 2% vs4-bromo-A23187: 13 +/- 4%; P = 0.03). NE treatment increased caspase-9 activity to 197 +/- 62% over control myocytes (P < 0.0001), whereas no caspase-8 activation was detectable. This increase in caspase-9 activity was blocked by isoflurane at 1.6 MAC and halothane at 1.2 MAC. CONCLUSIONS: Volatile anesthetics offer significant protection against beta-adrenergic apoptotic death signaling in ventricular cardiomyocytes. The authors present evidence that this protection is mainly mediated through modulation of cellular Ca2+ homeostasis and inhibition of the apoptosis initiator caspase-9.     

Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice

Drugs that suppress neuronal activity, including general anesthetics used in pediatric and obstetric medicine, trigger neuroapoptosis in the developing rodent brain. Exposure of infant rats for 6 hours to a combination of anesthetic drugs (midazolam, nitrous oxide, isoflurane) reportedly causes widespread apoptotic neurodegeneration, followed by lifelong cognitive deficits. Isoflurane, the dominant ingredient in this triple cocktail, has not been evaluated individually for apoptogenic potential. It was recently reported that (1) the minimum alveolar concentration (MAC) for anesthetizing infant mice with isoflurane is 2.26%, and; (2) that infant mice, without assisted respiration, maintain normal arterial oxygen values but become hypoglycemic when exposed to isoflurane 3% for 30 minutes, then 1.8% for 1 hour (1.46 MAC-hours). In the present experiments, infant mice were exposed to isoflurane at various sub-MAC concentrations and durations, and the brains were evaluated quantitatively 5 hours after initiation of anesthesia exposure to determine the number of neuronal profiles undergoing apoptosis. Blood glucose values were also determined under each of these conditions. All conditions tested (isoflurane at 0.75% for 4 h, 1.5% for 2 h, 2.0% for 1 h) triggered a statistically significant increase in neuroapoptosis compared with the rate of spontaneous apoptosis in littermate controls. Blood glucose determinations ruled out hypoglycemia as a potential cause of the brain damage. It is concluded that exposure to sub-MAC concentrations of isoflurane for one or more hours triggers neuroapoptosis in the infant mouse brain. These findings are consistent with other recent evidence demonstrating that brief exposure to ethanol, ketamine, or midazolam triggers neuroapoptosis in the developing mouse brain.     

Xenon Mitigates Isoflurane-induced Neuronal Apoptosis in the Developing Rodent Brain

Background: Anesthetics, including isoflurane and nitrous oxide, an antagonist of the N-methyl-d-aspartate subtype of the glutamate receptor, have been demonstrated to induce apoptotic neurodegeneration when administered during neurodevelopment. Xenon, also an N-methyl-d-aspartate antagonist, not only lacks the characteristic toxicity produced by other N-methyl-d-aspartate antagonists, but also attenuates the neurotoxicity produced by this class of agent. Therefore, the current study sought to investigate xenon's putative protective properties against anesthetic-induced neuronal apoptosis.

Method: Separate cohorts (n = 5 or 6 per group) of 7-day-old rats were randomly assigned and exposed to eight gas mixtures: air, 75% nitrous oxide, 75% xenon, 0.75% isoflurane, 0.75% isoflurane plus 35% or 75% nitrous oxide, 0.75% isoflurane plus 30% or 60% xenon for 6 h. Rats were killed, and cortical and hippocampal apoptosis was assessed using caspase-3 immunostaining. In separate cohorts, cortices were isolated for immunoblotting of caspase 3, caspase 8, caspase 9, and cytochrome c. Organotypic hippocampal slices of postnatal mice pups were derived and cultured for 24 h before similar gas exposures, as above, and subsequently processed for caspase-3 immunostaining.

Results: In vivo administration of isoflurane enhances neuronal apoptosis. When combined with isoflurane, nitrous oxide significantly increases whereas xenon significantly reduces apoptosis to a value no different from that of controls. In vitro studies corroborate the ability of xenon to attenuate isoflurane-induced apoptosis. Isoflurane enhanced expression of indicators of the intrinsic and common apoptotic pathways; this enhancement was increased by nitrous oxide but attenuated by xenon.     

Hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells: The effects of glutamate and protection by purines

Glutamate has toxic effects on a number of tissues, partly by inducing toxic (e.g., oxidative) stress, whereas adenosine can be protective. Since there is evidence that glutamate and adenosine receptors are present in bone, we set out to study whether oxidative stress, induced by hydrogen peroxide (H2O2), affected viability in the MC3T3-E1 osteoblast-like cell line and whether treatment with adenosine receptor ligands attenuated this. Hydrogen peroxide (100 microM to 5 mM) reduced the viability of the MC3T3-E1 cells, while catalase reversed this cell loss and itself had some mitogenic effect. Superoxide dismutase (SOD) increased the number of viable cells alone but failed to modify significantly the effect of H2O2 treatments. Glutamate (100 microM, 1 mM) and NMDA (10 microM), applied alone for up to 1 h, had a mitogenic effect (P < 0.05). Adenosine A1 and A2A receptor agonists and antagonists at low and high concentrations showed some mitogenic effects when added singly, but only high concentrations of the agonists showed significant protection against cell death resulting from H2O2 treatments. Contributions from both apoptotic and necrotic pathways were implicated in the H2O2-induced cell loss as was demonstrated by the use of the caspase-3 inhibitor (Z-DEVD-fmk) and the PARP-1 inhibitor (DPQ). The results demonstrate that hydrogen peroxide was toxic to MC3T3-E1 cells, whereas glutamate was not and may even have a trophic influence. Adenosine and its receptors afforded some protection to osteoblasts against cellular death mediated partly by apoptosis and partly by necrosis.     

Effects of Volatile Anesthetics on N-methyl-d-aspartate Excitotoxicity in Primary Rat Neuronal-Glial Cultures

Background: Volatile anesthetics are known to ameliorate experimental ischemic brain injury. A possible mechanism is inhibition of excitotoxic cascades induced by excessive glutamatergic stimulation. This study examined interactions between volatile anesthetics and excitotoxic stress.

Methods: Primary cortical neuronal-glial cultures were exposed to N-methyl-d-aspartate (NMDA) or glutamate and isoflurane (0.1-3.3 mm), sevoflurane (0.1-2.9 mm), halothane (0.1-2.9 mm), or 10 [mu]m (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801). Lactate dehydrogenase release was measured 24 h later. In other cultures, effects of volatile anesthetics on Ca++ uptake and mitochondrial membrane potential were determined in the presence or absence of NMDA (0-200 [mu]m).

Results: Volatile anesthetics reduced excitotoxin induced lactate dehydrogenase release by up to 52% in a dose-dependent manner. At higher concentrations, this protection was reversed. When corrected for olive oil solubility, the three anesthetics offered equivalent protection. MK-801 provided near-complete protection. Ca++ uptake was proportionally reduced with increasing concentrations of anesthetic but did not account for reversal of protection at higher anesthetic concentrations. Given equivalent NMDA-induced Ca++ loads, cells treated with volatile anesthetic had greater lactate dehydrogenase release than those left untreated. At protective concentrations, volatile anesthetics partially inhibited NMDA-induced mitochondrial membrane depolarization. At higher concentrations, volatile anesthetics alone were sufficient to induce mitochondrial depolarization.     

Cyclooxygenase-2 inhibition protects cultured cerebellar granule neurons from glutamate-mediated cell death

Primary insults to the brain can initiate glutamate release that may result in excitotoxicity followed by neuronal cell death. This secondary process is mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA receptors in vivo and requires new gene expression. Neuronal cyclooxygenase-2 (COX2) expression is upregulated following brain insults, via glutamatergic and inflammatory mechanisms. The products of COX2 are bioactive prostanoids and reactive oxygen species that may play a role in neuronal survival. This study explores the role of neuronal COX2 in glutamate excitotoxicity using cultured cerebellar granule neurons (day 8 in vitro). Treatment with excitotoxic concentrations of glutamate or kainate transiently induced COX2 mRNA (two- and threefold at 6 h, respectively, p < 0.05, Dunnett) and prostaglandin production (five- and sixfold at 30 min, respectively, p < 0.05, Dunnett). COX2 induction peaked at toxic concentrations of these excitatory amino acids. Surprisingly, NMDA, L-quisqualate, and trans-ACPD did not induce COX2 mRNA at any concentration tested. The glutamate receptor antagonist NBQX (5 microM, AMPA/kainate receptor) completely inhibited kainate-induced COX2 mRNA and partially inhibited glutamate-induced COX2 (p < 0.05, Dunnett). Other glutamate receptor antagonists, such as MK-801 (1 microM, NMDA receptor) or MCPG (500 microM, class 1 metabotropic receptors), partially attenuated glutamate-induced COX2 mRNA. These antagonists all reduced steady-state COX2 mRNA (p < 0.05, Dunnett). To determine whether COX2 might be an effector of excitotoxic cell death, cerebellar granule cells were pretreated (24 h) with the COX2-specific enzyme inhibitor, DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2((5)H)-furanone) prior to glutamate challenge. DFU (1 to 1000 nM) completely protected cultured neurons from glutamate-mediated neurotoxicity. Approximately 50% protection from NMDA-mediated neurotoxicity, and no protection from kainate-mediated neurotoxicity was observed. Therefore, glutamate-mediated COX2 induction contributes to excitotoxic neuronal death. These results suggest that glutamate, NMDA, and kainate neurotoxicity involve distinct excitotoxic pathways, and that the glutamate and NMDA pathways may intersect at the level of COX2.     

Adverse Effects of Methylene Blue on the Central Nervous System

Background: An increasing number of clinical observations suggest adverse neurologic outcome after methylene blue (MB) infusion in the setting of parathyroid surgery. Hence, the aim of the current study was to investigate the potentially neurotoxic effects of MB using a combination of in vivo and in vitro experimental approaches.

Methods: Isoflurane-anesthetized adult rats were used to evaluate the impact of a single bolus intravascular administration of MB on systemic hemodynamic responses and on the minimum alveolar concentration (MAC) of isoflurane using the tail clamp test. In vivo, MB-induced cell death was evaluated 24 h after MB administration using Fluoro-Jade B staining and activated caspase-3 immunohistochemistry. In vitro, neurotoxic effects of MB were examined in hippocampal slice cultures by measuring excitatory field potentials as well as propidium iodide incorporation after MB exposure. The impact of MB on dendritic arbor was evaluated in differentiated single cell neuronal cultures.

Results: Bolus injections of MB significantly reduced isoflurane MAC and initiated widespread neuronal apoptosis. Electrophysiologic recordings in hippocampal slices revealed a rapid suppression of evoked excitatory field potentials by MB, and this was associated with a dose-dependent effect of this drug on cell death. Dose-response experiments in single cell neuronal cultures revealed that a 2-h-long exposure to MB at non-cell-death-inducing concentrations could still induce significant retraction of dendritic arbor.     

Activity-dependent regulation of neuronal apoptosis in neonatal mouse cerebral cortex

A massive neuronal loss during early postnatal development has been well documented in the murine cerebral cortex, but the factors that drive cells into apoptosis are largely unknown. The role of neuronal activity in developmental apoptosis was studied in organotypic neocortical slice cultures of newborn mice. Multielectrode array and whole-cell patch-clamp recordings revealed spontaneous network activity characterized by synchronized burst discharges, which could be blocked by tetrodotoxin and ionotropic glutamate receptor antagonists. The identical neuropharmacological manipulations also caused a significant increase in the number of apoptotic neurons as early as 6 h after the start of drug treatment. Moreover, inhibition of the NMDA receptor subunit NR2A or NR2B induced a differential short-term versus delayed increase in the apoptosis rate, respectively. Activation of L-type, voltage-dependent calcium channels was neuroprotective and could prevent activity-dependent apoptosis during NMDA receptor blockade. Furthermore, this effect involved phosphorylation of cAMP response element-binding protein and activation of the tropomyosin-related kinase (Trk) receptors. Inhibition of electrical synapses and blockade of ionotropic gamma-aminobutyric acid receptors induced specific changes in spontaneous electrical activity patterns, which caused an increase in caspase-3-dependent cell death. Our results demonstrate that synchronized spontaneous network bursts activating ionotropic glutamate receptors promote neuronal survival in the neonatal mouse cerebral cortex.     

Cerebral cortical neuron apoptosis after mild excitotoxic injury in vitro: different roles of mesencephalic and cortical astrocytes

OBJECTIVE: Increasing evidence supports the presence of neuronal apoptosis after ischemic or excitotoxic brain injury. Astrocytes, which exhibit significant regional differences in function, may exert a protective effect on neurons exposed to ischemic injury. We examined the effects of astrocytes derived from different regions of the central nervous system on neuronal apoptosis after mild excitotoxic injury in tissue culture. METHODS: Purified astrocyte cultures derived from P4 rat cerebral cortex or mesencephalon showed transient cell swelling but no cell death when exposed to 50 micromol/L glutamate for 5 minutes. When mixed neuronal/glial cocultures were exposed to the same glutamate dose, neuron death was observed. Necrotic and apoptotic cell death during 24 hours was examined using morphological criteria, nuclear staining, triphosphate nick end labeling, and trypan blue exclusion. RESULTS: We found that cortical neurons that elaborate a more extensive dendritic arbor when grown on homotypic astrocytes are more likely to undergo apoptosis than neurons with a limited dendritic arbor grown on heterotypic astrocytes. By contrast, a similar number of neurons undergo necrotic cell death. CONCLUSION: This finding may be associated with 1) increased vulnerability of neurons with a more elaborate dendrite structure to mild excitotoxic injury, or 2) regional differences in the ability of astrocytes to attenuate apoptosis.     

The dose-dependent effects of isoflurane on outcome from severe forebrain ischemia in the rat

Isoflurane improves outcome against cerebral ischemia in the rat. However, the optimal neuroprotective concentration has not been defined. We examined the effects of different isoflurane concentrations on outcome from severe forebrain ischemia in the rat. Fasted rats were subjected to 0.5, 1.0, 1.5, 2.0, or 2.5 minimum alveolar concentration (MAC) isoflurane during 10 min bilateral carotid occlusion plus systemic hypotension. Each isoflurane concentration was administered only before ischemia. Arterial blood pressure was not pharmacologically manipulated. After ischemia, the anesthetic regimen was changed to fentanyl/nitrous oxide and maintained for 2 h. Pericranial temperature was maintained normothermic during the experiment. Neuromotor score, % dead hippocampal CA1 neurons, and cortical injury were measured 5 days postischemia. Preischemic arterial blood pressure decreased as MAC was increased. Animals administered >1.0 MAC frequently exhibited postischemic seizures resulting in increased mortality. There was no difference among MAC conditions for % dead CA1 neurons (93 approximately 95%). In the cortex, neuronal necrosis was less severe with 0.5 MAC and 1.0 MAC isoflurane relative to >1.0 MAC values. The neuromotor score in the 1.0 MAC isoflurane group was superior to the 2.5 MAC group. Dose-dependent effects of preischemic administration of isoflurane on histologic and behavioral outcome after severe forebrain ischemia were observed. Isoflurane MAC values <1.5 provided superior overall outcome relative to larger isoflurane concentrations.     

Rapid subcellular redistribution of Bax precedes caspase-3 and endonuclease activation during excitotoxic neuronal apoptosis in rat brain

Neuronal apoptosis is induced prominently in the newborn rodent brain by glutamate receptor excitotoxicity and related insults, including trauma and hypoxia-ischemia. However, the molecular mechanisms of this neurodegeneration are unclear. We tested the hypothesis that changes in the subcellular distribution of the proapoptotic protein Bax precede the activation of downstream apoptosis-effector mechanisms such as caspase-3 cleavage and endonuclease activation during the progression of excitotoxic neuronal apoptosis in the striatum of newborn rat. Kainic acid (4 nmol) was injected into striatum of anesthetized 7-day-old rats, and the animals were killed at 2, 6, 12, and 24 h postinsult. Controls were age-matched, vehicle-injected, or naive rats. Counts of ultrastructurally confirmed striatal neuron apoptosis in brain sections were highest at 24 h. Striatal tissue was microdissected and fractionated into cytosolic, mitochondrial-, and nuclear-enriched compartments. Immunoblots showed that Bax translocates from the cytosol fraction to the mitochondrial fraction, with maximal translocation by 2 h in the absence of changes in mitochondrial accumulation. Cleaved caspase-3 levels increase progressively in both cytosolic and mitochondrial fractions between 6 and 24 h. Cleaved caspase-3 accumulates in apoptotic striatal neurons as shown by immunolocalization. Internucleosomal fragmentation of DNA coincides with caspase-3 cleavage. We conclude that rapid translocation of Bax to mitochondria precedes caspase-3 and endonuclease activation during excitotoxic neuronal apoptosis in newborn rat brain and that initiation of this death cascade occurs within 2 h after glutamate receptor activation.     

Modulation of NMDA receptor function by ketamine and magnesium. Part II: interactions with volatile anesthetics

Mg2+ and ketamine interact superadditively at N- methyl-D-aspartate (NMDA) receptors, which may explain the clinical efficacy of the combination. Because patients are usually exposed concomitantly to volatile anesthetics, we tested the hypothesis that volatile anesthetics interact with ketamine and/or Mg2+ at recombinantly expressed NMDA receptors. NR1/NR2A or NR1/NR2B receptors were expressed in Xenopus oocytes. We determined the effects of isoflurane, sevoflurane, and desflurane on NMDA receptor signaling, alone and in combination with S(+)-ketamine (4.1 microM on NR1/NR2A, 3.0 microM on NR2/NR2B) and/or Mg2+ (416 microM on NR1/NR2A, 629 microM on NR1/NR2B). Volatile anesthetics inhibited NR1/NR2A and NR1/NR2B glutamate receptor function in a reversible, concentration-dependent, voltage-insensitive and noncompetitive manner (half-maximal inhibitory concentration at NR1/NR2A receptors: 1.30 +/- 0.02 minimum alveolar anesthetic concentration [MAC] for isoflurane, 1.18 +/- 0.03 MAC for desflurane, 1.24 +/- 0.06 MAC for sevoflurane; at NR1/NR2B receptors: 1.33 +/- 0.12 MAC for isoflurane, 1.22 +/- 0.08 MAC for desflurane, and 1.28 +/- 0.08 MAC for sevoflurane). On both NR1/NR2A and NR1/NR2B receptors, 50% inhibitory concentration for volatile anesthetics was reduced approximately 20% by Mg2+, approximately 30% by S(+)-ketamine, and approximately 50% by the compounds in combination. Volatile anesthetic effects on NMDA receptors can be potentiated significantly by Mg2+, S(+)-ketamine, or-most profoundly-both. Therefore, the analgesic effects of ketamine and Mg2+, are likely to be enhanced in the presence of volatile anesthetics. IMPLICATIONS: Clinically relevant concentrations of volatile anesthetics inhibit functioning of N-methyl-D-aspartate receptors expressed recombinantly in Xenopus oocytes. This inhibition is reversible, concentration-dependent and voltage-insensitive, and results from noncompetitive antagonism of glutamate/glycine signaling. In addition, these effects can be potentiated significantly by co-application of either Mg2+, S(+)-ketamine, or--most profoundly--both.     

Sensitivity of the alpha7 nicotinic acetylcholine receptor to isoflurane may depend on receptor inactivation

In previous studies, we demonstrated that nicotinic acetylcholine receptors (nAChRs) composed of the alpha7 subunit are unaffected by the co-application of isoflurane with agonists at concentrations up to 640 microM (two times the minimum alveolar anesthetic concentration). Modulation of alpha7-nAChR activity by isoflurane might have important behavioral ramifications because these receptors are expressed diffusely in the central and peripheral nervous systems and play pre- and postsynaptic roles in synaptic transmission. Here we have demonstrated that under some potentially physiologically relevant circumstances, the activation of alpha7 nAChRs may be inhibited by clinically relevant concentrations of isoflurane. We evaluated isoflurane inhibition of alpha7 nAChRs from chicks and humans expressed in Xenopus oocytes using two-electrode voltage clamp methodology. We determined the influence of time of preperfusion of isoflurane, agonist concentration, and membrane potential on inhibition by isoflurane. Both activation by a large concentration of agonist and isoflurane preperfusion increased inhibition. The half-maximal inhibitory concentration for isoflurane inhibition of chick alpha7 nAChR with isoflurane preperfusion and activation by 100 microM of acetylcholine was 938 +/- 26, and when activated by 1 mM of acetylcholine, it was 408 +/- 51 microM. The increase in inhibition with isoflurane preexposure and large agonist concentration raises the possibility that isoflurane interacts preferentially with a closed or closed-desensitized state of the channel. IMPLICATIONS: Nicotinic receptors expressed in the brain have been considered a possible target for the actions of isoflurane. We studied the effect of isoflurane on alpha7 type nicotinic receptors expressed in Xenopus oocytes. We find that when activated by large concentrations of acetylcholine, alpha7 nicotinic receptors are inhibited by isoflurane at concentrations near MAC.     

Subunit-dependent inhibition of human neuronal nicotinic acetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine and dizocilpine

Background The neuronal mechanisms responsible for dissociative anesthesia remain controversial. N-methyl-D-aspartate (NMDA) receptors are inhibited by ketamine and related drugs at concentrations lower than those required for anesthetic effects. Thus, the authors studied whether ligand-gated ion channels other than NMDA receptors might display a sensitivity to ketamine and dizocilpine that is consistent with concentrations required for anesthesia. METHODS: Heteromeric human neuronal nicotinic acetylcholine receptors (hnAChR channels alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2 and alpha4beta4), 5-hydroxytryptamine3 (5-HT3), alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) and alpha1 glycine receptors were expressed in Xenopus oocytes, and effects of ketamine and dizocilpine were studied using the two-electrode voltage-clamp technique. RESULTS: Both ketamine and dizocilpine inhibited hnAChRs in a noncompetitive and voltage-dependent manner. Receptors containing beta1 subunits were more sensitive to ketamine and dizocilpine than those containing beta2 subunits. The inhibitor concentration for half-maximal response (IC50) values for ketamine of hnAChRs composed of beta4 subunits were 9.5-29 microM, whereas those of beta2 subunits were 50-92 microM. Conversely, 5-HT3 receptors were inhibited only by concentrations of ketamine and dizocilpine higher than the anesthetic concentrations. This inhibition was mixed (competitive/noncompetitive). GABAA and glycine receptors were very resistant to dissociative anesthetics. CONCLUSIONS: Human nAChRs are inhibited by ketamine and dizocilpine at concentrations possibly achieved in vivo during anesthesia in a subunit-dependent manner, with beta subunits being more critical than alpha subunits. Conversely, 5-HT3, GABAA, and glycine receptors were relatively insensitive to dissociative anesthetics.