Isoflurane prevents delayed cell death in an organotypic slice culture model of cerebral ischemia.

BACKGROUND: General anesthetics reduce neuronal death caused by focal cerebral ischemia in rodents and by in vitro ischemia in cultured neurons and brain slices. However, in intact animals, the protective effect may enhance neuronal survival for only several days after an ischemic injury, possibly because anesthetics prevent acute but not delayed cell death. To further understand the mechanisms and limitations of volatile anesthetic neuroprotection, the authors developed a rat hippocampal slice culture model of cerebral ischemia that permits assessment of death and survival of neurons for at least 2 weeks after simulated ischemia. METHODS: Survival of CA1, CA3, and dentate gyrus neurons in cultured hippocampal slices (organotypic slice culture) was examined 2-14 days after 45 min of combined oxygen-glucose deprivation at 37 degrees C (OGD). Delayed cell death was serially measured in each slice by quantifying the binding of propidium iodide to DNA with fluorescence microscopy. RESULTS: Neuronal death was greatest in the CA1 region, with maximal death occurring 3-5 days after OGD. In CA1, cell death was 80 +/- 18% (mean +/- SD) 3 days after OGD and was 80-100% after 1 week. Death of 70 +/- 16% of CA3 neurons and 48 +/- 28% of dentate gyrus neurons occurred by the third day after OGD. Both isoflurane (1%) and the N-methyl-D-aspartate antagonist MK-801 (10 microm) reduced cell death to levels similar to controls (no OGD) for 14 days after the injury. Isoflurane also reduced cell death in CA1 and CA3 caused by application of 100 but not 500 microm glutamate. Cellular viability (calcein fluorescence) and morphology were preserved in isoflurane-protected neurons. CONCLUSIONS: In an in vitro model of simulated ischemia, 1% isoflurane is of similar potency to 10 microm MK-801 in preventing delayed cell death. Modulation of glutamate excitotoxicity may contribute to the protective mechanism.     

Isoflurane Prevents Delayed Cell Death in an Organotypic Slice Culture Model of Cerebral Ischemia

Background: General anesthetics reduce neuronal death caused by focal cerebral ischemia in rodents and by in vitro ischemia in cultured neurons and brain slices. However, in intact animals, the protective effect may enhance neuronal survival for only several days after an ischemic injury, possibly because anesthetics prevent acute but not delayed cell death. To further understand the mechanisms and limitations of volatile anesthetic neuroprotection, the authors developed a rat hippocampal slice culture model of cerebral ischemia that permits assessment of death and survival of neurons for at least 2 weeks after simulated ischemia.

Methods: Survival of CA1, CA3, and dentate gyrus neurons in cultured hippocampal slices (organotypic slice culture) was examined 2-14 days after 45 min of combined oxygen-glucose deprivation at 37[degrees]C (OGD). Delayed cell death was serially measured in each slice by quantifying the binding of propidium iodide to DNA with fluorescence microscopy.

Results: Neuronal death was greatest in the CA1 region, with maximal death occurring 3-5 days after OGD. In CA1, cell death was 80 +/- 18% (mean +/- SD) 3 days after OGD and was 80-100% after 1 week. Death of 70 +/- 16% of CA3 neurons and 48 +/- 28% of dentate gyrus neurons occurred by the third day after OGD. Both isoflurane (1%) and the N-methyl-d-aspartate antagonist MK-801 (10 [mu]m) reduced cell death to levels similar to controls (no OGD) for 14 days after the injury. Isoflurane also reduced cell death in CA1 and CA3 caused by application of 100 but not 500 [mu]m glutamate. Cellular viability (calcein fluorescence) and morphology were preserved in isoflurane-protected neurons.     

Volatile anesthetics increase intracellular calcium in cerebrocortical and hippocampal neurons

BACKGROUND: An increase in intracellular calcium concentration ([Ca2+]i) in neurons has been proposed as an important effect of volatile anesthetics, because they alter signaling pathways that influence neurotransmission. However, the existing data for anesthetic-induced increases in [Ca2+]i conflict. METHODS: Changes in [Ca2+]i were measured using fura-2 fluorescence spectroscopy in rat cortical brain slices at 90, 185, 370, and 705 microM isoflurane. To define the causes of an increase in [Ca2+]i, slices were studied in Ca2+-free medium, in the presence of Ca2+-channel blockers, and in the presence of the Ca2+-release inhibitor azumolene. The authors compared the effect of the volatile anesthetic with that of the nonanesthetic compound 1,2-dichlorohexafluorocyclobutane. Single-dose experiments in CA1 neurons in hippocampal slices with halothane (360 microM) and in acutely dissociated CA1 neurons with halothane (360 microM) and isoflurane (445 microM) also were performed. RESULTS: Isoflurane at 0.5, 1, and 2 minimum alveolar concentrations increased basal [Ca2+]i in cortical slices in a dose-dependent manner (P < 0.05). This increase was not altered by Ca2+-channel blockers or Ca2+-free medium but was reduced 85% by azumolene. The nonanesthetic 1,2-dichlorohexafluorocyclobutane did not increase [Ca2+]i. In dissociated CA1 neurons, isoflurane reversibly increased basal [Ca2+]i by 15 nM (P < 0.05). Halothane increased [Ca2+]i in dissociated CA1 neurons and CA1 neurons in hippocampal slices by approximately 30 nM (P < 0.05). CONCLUSIONS: (1) Isoflurane and halothane reversibly increase [Ca2+]i in isolated neurons and in neurons within brain slices. (2) The increase in [Ca2+]i is caused primarily by release from intracellular stores. (3) Increases in [Ca2+]i occur with anesthetics but not with the nonanesthetic 1,2-dichlorohexafluorocyclobutane.     

Thiopental Inhibits Increases in [Ca2+]i Induced by Membrane Depolarization, NMDA Receptor Activation, and Ischemia in Rat Hippocampal and Cortical Slices

Background: This study examined the effects of thiopental on intracellular calcium ([Ca2+]i) changes induced by membrane depolarization, N-methyl-D-aspartate (NMDA) receptor activation, and ischemia.

Methods: Experiments were performed in brain slices prepared from Wistar rats. [Ca2+]i measurements were taken on the CA1 pyramidal cell layer of the hippocampus or layers II to III of the somatosensory cortex using the fura-2 fluorescence technique. Membrane depolarization and NMDA receptor activation were induced by exposing slices to 60 mM K+ and 100 [micro sign]M NMDA, respectively. In vitro ischemia was induced by superfusing slices with glucose-free Krebs solution equilibrated with 95% nitrogen and 5% carbon dioxide. Thiopental was applied 5 min before application of high K+ and NMDA, or before in vitro ischemia.

Results: Ischemia for 15 min produced a characteristic [Ca2+]i increase in both hippocampal and cortical slices. Thiopental prolonged the latency to the appearance of the [Ca2+]i plateau and reduced the magnitudes of increase in [Ca2+]i 8, 10, and 15 min after the onset of ischemia. Thiopental also suppressed the high K+-and NMDA-induced [Ca (2+)]i increases. The NMDA-induced [Ca2+]i increases were attenuated to a greater extent in cortical slices than were those in hippocampal slices. The inhibition of thiopental on the 200-[micro sign]M NMDA-mediated [Ca2+]i response was confirmed in cultured cortical neurons.     

Anesthetics and mild hypothermia similarly prevent hippocampal neuron death in an in vitro model of cerebral ischemia

BACKGROUND: General anesthetics reduce neuron loss following focal cerebral ischemia in rodents. The relative efficacy of this action among different anesthetics clinically used for neuroprotection is uncertain. In addition, it remains unclear how anesthetics compare to neuroprotection afforded by mild hypothermia. This study was performed to evaluate the comparative effects of isoflurane, sodium pentothal, and mild hypothermia in a hippocampal slice model of cerebral ischemia and to determine if the mechanism of neuroprotection of isoflurane involves inhibition of glutamate excitotoxicity. METHODS: Survival and morphology of CA1, CA3, and dentate gyrus neurons in rat hippocampal slices were examined after 10 or 20 min of combined oxygen-glucose deprivation (in vitro ischemia) followed by a 5-h recovery period. RESULTS: 10 or 20 min in vitro ischemia at 37 degrees C killed 35-40% of neurons in CA1 (P < 0.001), 6% in CA3 (not significant) and 18% in dentate (P < 0.05). Isoflurane (0.7 and 2.0%, approximately 0.45 and 1.5 minimum alveolar concentration), pentothal (50 microm, approximately 1 minimum alveolar concentration equivalent) and mild hypothermia (34 degrees C) all reduced CA1 cell loss and morphologic damage to similar degrees in 10- and 20-min periods of ischemia (P < 0.001). The noncompetitive N-methyl-D-aspartate antagonist MK-801 prevented cell damage, showing that N-methyl-D-aspartate receptor activation is an important mechanism of injury in this model. Glutamate (1 mm) produced cell loss similar to in vitro ischemia. Isoflurane (2%) prevented cell damage from glutamate exposure. CONCLUSIONS: In hippocampal slices, neuron death from simulated ischemia was predominately due to activation of glutamate receptors. Isoflurane, sodium pentothal, an N-methyl-D-aspartate receptor antagonist, and mild hypothermia prevented cell death to similar degrees. For isoflurane, the mechanism appears to involve attenuation of glutamate excitotoxicity.     

gamma-Aminobutyric acid-A receptors contribute to isoflurane neuroprotection in organotypic hippocampal cultures

The mechanisms by which anesthetics such as isoflurane reduce cell death in rodent models of cerebral ischemia remain incompletely defined. Reduction in glutamate excitotoxicity explains some but not all of isoflurane's neuroprotection. Because isoflurane potentiates gamma-aminobutyric acid (GABA) receptor-mediated ion fluxes and GABA(A) receptor agonists have neuroprotective effects, we hypothesized that GABA(A) receptors contribute to isoflurane neuroprotection. As a model of cerebral ischemia and recovery, we used rat hippocampal slice cultures. Survival of CA1, CA3, and dentate neurons was examined 2 and 3 days after 1-h combined oxygen-glucose deprivation (OGD) at 37 degrees C. To define the role of GABA(A) receptors in mediating protection, the effect of 1% isoflurane on cell survival was examined in the presence of the GABA(A) antagonist bicuculline during OGD. Cell death was measured with propidium iodide fluorescence. Isoflurane and the selective GABA(A) agonist muscimol (25 micro M) reduced cell death after OGD to values similar to slices not exposed to OGD, with the exception that muscimol did not reduce cell death in CA3 neurons 2 days after OGD. The GABA(A) antagonist bicuculline reduced the neuroprotective effects of isoflurane on hippocampal neurons 2 and 3 days after OGD. We conclude that GABA(A) receptors contribute to neuroprotection against OGD produced by isoflurane in the hippocampal slice model. Based on this and other studies, it is likely that neuroprotection produced by isoflurane is multifactorial and includes actions at both GABA(A) and glutamate receptors and possibly other mechanisms. IMPLICATIONS: Isoflurane is neuroprotective in rodent brain ischemia models, but the mechanisms for this effect remain incompletely defined. In organotypic cultures of rat hippocampus, we show that protection of CA1, CA3, and dentate neurons by 1% isoflurane from death caused by oxygen and glucose deprivation involves GABA(A) receptors.     

Anesthetics and Mild Hypothermia Similarly Prevent Hippocampal Neuron Death in an In Vitro Model of Cerebral Ischemia

Background: General anesthetics reduce neuron loss following focal cerebral ischemia in rodents. The relative efficacy of this action among different anesthetics clinically used for neuroprotection is uncertain. In addition, it remains unclear how anesthetics compare to neuroprotection afforded by mild hypothermia. This study was performed to evaluate the comparative effects of isoflurane, sodium pentothal, and mild hypothermia in a hippocampal slice model of cerebral ischemia and to determine if the mechanism of neuroprotection of isoflurane involves inhibition of glutamate excitotoxicity.

Methods: Survival and morphology of CA1, CA3, and dentate gyrus neurons in rat hippocampal slices were examined after 10 or 20 min of combined oxygen-glucose deprivation (in vitro ischemia) followed by a 5-h recovery period.

Results: 10 or 20 min in vitro ischemia at 37[degrees]C killed 35-40% of neurons in CA1 (P < 0.001), 6% in CA3 (not significant) and 18% in dentate (P < 0.05). Isoflurane (0.7 and 2.0%, [almost equal to] 0.45 and 1.5 minimum alveolar concentration), pentothal (50 [mu]m, [almost equal to] 1 minimum alveolar concentration equivalent) and mild hypothermia (34[degrees]C) all reduced CA1 cell loss and morphologic damage to similar degrees in 10- and 20-min periods of ischemia (P < 0.001). The noncompetitive N-methyl-d-aspartate antagonist MK-801 prevented cell damage, showing that N-methyl-d-aspartate receptor activation is an important mechanism of injury in this model. Glutamate (1 mm) produced cell loss similar to in vitro ischemia. Isoflurane (2%) prevented cell damage from glutamate exposure.     

The effects of thiopental and propofol on cell swelling induced by oxygen/glucose deprivation in the CA1 pyramidal cell layer of rat hippocampal slices

Cellular swelling has been implicated as an early process after cerebral ischemia. We compared the effects of two commonly used IV anesthetics, thiopental and propofol, on hippocampal CA1 pyramidal cell swelling induced by oxygen/glucose deprivation (OGD) in vitro. Experiments were performed in rat hippocampal slices. Cell swelling in the CA1 pyramidal cell layer was evaluated by determining light transmittance (LT) change through the slices and by histopathological examination. For LT experiments, OGD was induced for 10 min by superfusing slices with glucose-free artificial cerebrospinal fluid equilibrated with 95% nitrogen and 5% CO(2). Thiopental and propofol were present 10 min before and during the period of OGD. The results showed that thiopental (100 and 400 microM), but not propofol (40 and 160 microM), significantly prolonged latency to the peak of LT increase after the onset of OGD. Consistent with the LT experiments, histopathological examination revealed that thiopental, but not propofol, attenuated CA1 pyramidal cell expansion and the gap diminution between CA1 pyramidal cells induced by OGD. These results suggest that thiopental, but not propofol, reduces the neuronal cell swelling caused by OGD. Whether the reduction of cell swelling is related to reduction in cell injury caused by OGD remains to be investigated. IMPLICATIONS: We demonstrated that thiopental, but not propofol, attenuates ischemic neuronal swelling induced by oxygen/glucose deprivation in an in vitro ischemic model.     

NMDA receptors in cortical development are essential for the generation of coordinated increases in [Ca2+](i) in "neuronal domains"

Spontaneous correlated activity regulates the precision of developing neural circuits. A synchronized elevation of intracellular calcium ion concentration, [Ca(2+)](i), occurred in 5-50 adjacent neurons--known as a "neuronal domain"--in developing neocortex. This coordinated response of neuronal cells is mediated by the diffusion of inositol trisphosphate (IP(3)) via gap-junction channels. In this study, we utilized the N-methyl-D-aspartate (NMDA)-type glutamate receptor epsilon 2 (GluR epsilon 2/NR2B)(-/-) mouse, which does not possess any functional NMDA receptors in the developing neocortex, and showed that NMDA receptors are essential for the generation of "neuronal domains". First, the frequency of spontaneously occurring neuronal domains in brain slices from GluR epsilon 2(-/-) mice was significantly reduced compared to that seen in brain slices from wild-type mice. Secondly, IP(3) injection into a single neuron in a cortical slice from a GluR epsilon 2(-/-) brain resulted in very few neuronal domains being observed, but an injection similarly made into a neuron in a wild-type slice promptly resulted in neuronal domains. Even in the GluR epsilon 2(-/-) brain, the elevation of intracellular [Ca2+](i) was observed frequently in single neurons and microinjection of IP(3) produced an elevation of [Ca2+](i) in the injected cells. These results suggest that the diffusion of IP(3) into the surrounding neurons via gap junctions is almost completely absent in the GluR epsilon 2(-/-) brain. Our results may reflect the critical role of NMDA receptors in the formation of cortical circuitry, probably via the regulation of gap-junction channels between immature cortical neurons.     

Volatile Anesthetics Increase Intracellular Calcium in Cerebrocortical and Hippocampal Neurons

Background: An increase in intracellular calcium concentration ([Ca2+]i) in neurons has been proposed as an important effect of volatile anesthetics, because they alter signaling pathways that influence neurotransmission. However, the existing data for anesthetic-induced increases in [Ca2+]i conflict.

Methods: Changes in [Ca2+]i were measured using fura-2 fluorescence spectroscopy in rat cortical brain slices at 90, 185, 370, and 705 [micro sign]M isoflurane. To define the causes of an increase in [Ca2+]i, slices were studied in Ca2+-free medium, in the presence of Ca2+-channel blockers, and in the presence of the Ca2+-release inhibitor azumolene. The authors compared the effect of the volatile anesthetic with that of the nonanesthetic compound 1,2-dichlorohexafluorocyclobutane. Single-dose experiments in CA1 neurons in hippocampal slices with halothane (360 [micro sign]M) and in acutely dissociated CA1 neurons with halothane (360 [micro sign]M) and isoflurane (445 [micro sign]M) also were performed.

Results: Isoflurane at 0.5, 1, and 2 minimum alveolar concentrations increased basal [Ca2+]i in cortical slices in a dose-dependent manner (P < 0.05). This increase was not altered by Ca2+-channel blockers or Ca2+-free medium but was reduced 85% by azumolene. The nonanesthetic 1,2-dichlorohexafluorocyclobutane did not increase [Ca2+] (i). In dissociated CA1 neurons, isoflurane reversibly increased basal [Ca (2+)]i by 15 nM (P < 0.05). Halothane increased [Ca2+]i in dissociated CA1 neurons and CA1 neurons in hippocampal slices by approximately 30 nM (P < 0.05).     

Does propofol enhance GABA-mediated inhibition?

The effects of propofol on synaptic transmission were characterized and compared with pentobarbital in the rat hippocampal slice preparation. Hippocampal CA1 population spike after stimulation of Schaffer collaterals indicated that the postsynaptic response was primarily mediated by non-N-methyl-D-aspartate class glutamate receptors since it was abolished by the presence of 6,7-dinitroquinoxaline-2,3-dione (DNQX). Propofol and pentobarbital depressed CA1 population spike amplitude in a dose dependent fashion. Dose-response curves for population spike amplitudes were determined for propofol and pentobarbital and the concentrations producing a half-maximum response (ED(50)) were 110 microM and 160 microM for propofol and pentobarbital, respectively. By contrast, when GABA A-mediated inhibition was blocked by addition of 100 microM pictotoxin, propofol, in concentrations up to 400 microM had no significant effect on population spike amplitudes. These results suggest that propofol attenuates synaptic transmission in the central nervous system in part by enhancing GABA A-mediated inhibition and not by depressing glutamate-mediated excitation, as occurs with pentobarbital.     

Isoflurane Neuroprotection in Hypoxic Hippocampal Slice Cultures Involves Increases in Intracellular Ca2+ and Mitogen-activated Protein Kinases

Background: The volatile anesthetic isoflurane reduces acute and delayed neuron death in vitro models of brain ischemia, an action that the authors hypothesize is related to moderate increases in intracellular calcium concentration ([Ca2+]i). Specifically, the authors propose that during hypoxia, moderate increases in [Ca2+]i in the presence of isoflurane stimulates the Ca2+-dependent phosphorylation of members of the mitogen-activated protein kinase (MAP) kinase Ras-Raf-MEK-ERK pathway that are critical for neuroprotective signaling and suppression of apoptosis.

Methods: Death of CA1, CA3, and dentate neurons in rat hippocampal slice cultures was assessed by propidium iodide fluorescence 48-72 h after 60-75 min of hypoxia. [Ca2+]i in CA1 neurons was measured with fura-2 and fura-2 FF. Concentrations of the survival-signaling proteins Ras, MEK, MAP kinase p42/44, and protein kinase B (Akt) were assessed by immunostaining, and specific inhibitors were used to ascertain the role of Ca2+ and MAP kinases in mediating survival.

Results: Isoflurane, 1%, decreased neuron death in CA1, CA3, and dentate gyrus neurons after 60 but not 75 min of hypoxia. Survival of CA1 neurons required an inositol triphosphate receptor-dependent increase in [Ca2+]i of 30-100 nm that activated the Ras-Raf-MEK-ERK (p44/42) signaling pathway. Isoflurane also increased the phosphorylation of Akt during hypoxia.     

Activations of nPKCepsilon and ERK1/2 were involved in oxygen-glucose deprivation-induced neuroprotection via NMDA receptors in hippocampal slices of mice

Accumulated reports have suggested that activation of protein kinase C (PKC) isoforms may involve the activation of extracellular signal-regulated kinases (ERKs) in the neuronal response to ischemic/hypoxic stimuli. We have previously demonstrated that the membrane translocation of novel PKC (nPKC) epsilon increased in the early phase of cerebral ischemic/hypoxic preconditioning of mice. In this study, we used Western blot analysis and propidium iodide stain to determine whether the activations of nPKCepsilon and ERKs were involved in oxygen-glucose deprivation (OGD)-induced neuroprotection via N-methyl-D-aspartate (NMDA) receptors. The hippocampal slices of mice were exposed to OGD for 10 (OGD10) or 45 minutes (OGD45) to mimic mild (causing ischemic/hypoxic preconditioning) and severe (causing severe OGD) ischemia/hypoxia, respectively. We found that OGD10-induced nPKCepslilon membrane translocation was mediated by NMDA receptors, and both OGD10 and NMDA (1 microM, 30 min) pretreatment could protect Cornu Ammonis region 1 neurons against the subsequent severe OGD45. In addition, nPKCepsilon translocation inhibitor, epsilonV1-2 (1 microM, 30 min), and ERKs upstream mitogen-activated protein/extracellular signal regulated kinase kinase inhibitor, PD-98059 (20 microM, 30 min), could significantly inhibit OGD10 and NMDA-induced neuroprotection. These results suggest that OGD10-induced neuroprotection against severe OGD45 in the Cornu Ammonis region 1 region of the hippocampal slices was mediated by the activations of NMDA receptors, nPKCepsilon, and the downstream ERKs.     

Isoflurane neuroprotection in hypoxic hippocampal slice cultures involves increases in intracellular Ca2+ and mitogen-activated protein kinases

BACKGROUND: The volatile anesthetic isoflurane reduces acute and delayed neuron death in vitro models of brain ischemia, an action that the authors hypothesize is related to moderate increases in intracellular calcium concentration ([Ca2+]i). Specifically, the authors propose that during hypoxia, moderate increases in [Ca2+]i in the presence of isoflurane stimulates the Ca2+-dependent phosphorylation of members of the mitogen-activated protein kinase (MAP) kinase Ras-Raf-MEK-ERK pathway that are critical for neuroprotective signaling and suppression of apoptosis. METHODS: Death of CA1, CA3, and dentate neurons in rat hippocampal slice cultures was assessed by propidium iodide fluorescence 48-72 h after 60-75 min of hypoxia. [Ca2+]i in CA1 neurons was measured with fura-2 and fura-2 FF. Concentrations of the survival-signaling proteins Ras, MEK, MAP kinase p42/44, and protein kinase B (Akt) were assessed by immunostaining, and specific inhibitors were used to ascertain the role of Ca2+ and MAP kinases in mediating survival. RESULTS: Isoflurane, 1%, decreased neuron death in CA1, CA3, and dentate gyrus neurons after 60 but not 75 min of hypoxia. Survival of CA1 neurons required an inositol triphosphate receptor-dependent increase in [Ca2+]i of 30-100 nm that activated the Ras-Raf-MEK-ERK (p44/42) signaling pathway. Isoflurane also increased the phosphorylation of Akt during hypoxia. CONCLUSIONS: Isoflurane stimulates the phosphorylation of survival signaling proteins in hypoxic neurons. The mechanism involves a moderate increase in [Ca2+]i from release of Ca from inositol triphosphate receptor-dependent intracellular stores. The increase in [Ca2+]i sets in motion signaling via Ras and the MAP kinase p42/44 pathway and the antiapoptotic factor Akt. Isoflurane neuroprotection thus involves intracellular signaling well known to suppress both excitotoxic and apoptotic/delayed cell death.     

Neuroprotective effects of propofol in models of cerebral ischemia: inhibition of mitochondrial swelling as a possible mechanism

BACKGROUND: Propofol (2,6-diisopropylphenol) has been shown to attenuate neuronal injury in a number of experimental conditions, but studies in models of cerebral ischemia have yielded conflicting results. Moreover, the mechanisms involved in its neuroprotective effects are yet unclear. METHODS: The authors evaluated the neuroprotective effects of propofol in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. To investigate its possible mechanism of action, the authors then examined whether propofol could reduce Ca2+-induced rat brain mitochondrial swelling, an index of mitochondrial membrane permeability, as well as the mitochondrial swelling evoked by oxygen-glucose deprivation in CA1 pyramidal cells by transmission electron microscopy. Finally, they evaluated whether propofol could attenuate the infarct size and improve the neurobehavioral outcome in rats subjected to permanent middle cerebral artery occlusion in vivo. RESULTS: When present in the incubation medium during oxygen-glucose deprivation and the subsequent 24 h recovery period, propofol (10-100 microM) attenuated CA1 injury in hippocampal slices in vitro. Ca2+-induced brain mitochondrial swelling was prevented by 30-100 microM propofol, and so were the ultrastructural mitochondrial changes in CA1 pyramidal cells exposed to oxygen-glucose deprivation. Twenty-four hours after permanent middle cerebral artery occlusion, propofol (100 mg/kg, intraperitoneal) reduced the infarct size by approximately 30% when administered immediately after and up to 30 min after the occlusion. Finally, propofol administered within 30 min after middle cerebral artery occlusion was unable to affect the global neurobehavioral score but significantly preserved spontaneous activity in ischemic rats. CONCLUSIONS: These results show that propofol, at clinically relevant concentrations, is neuroprotective in models of cerebral ischemia in vitro and in vivo and that it may act by preventing the increase in neuronal mitochondrial swelling.     

Differential inhibitory effects of thiopental, thiamylal and phenobarbital on both voltage-gated calcium channels and NMDA receptors in rat hippocampal slices

Although it is known that there are some pharmacological differences between the structurally similar barbiturates, the underlying mechanism of action remains unclear. We have compared the effects of thiopental, thiamylal and phenobarbital on both voltage-gated calcium channels (VGCC) and N-methyl-D-aspartate (NMDA) receptors in rat hippocampal slices by determining changes in intracellular calcium ([Ca2+]i). Experiments were performed in adult rat hippocampal slices perfused with Krebs solution (37 degrees C). Concentrations of [Ca2+]i in the pyramidal cell layer of the CA1 region were measured using a calcium indicator dye, fura-2. To activate VGCC and NMDA receptors, slices were exposed to K+ 60 mmol litre-1 (< or = 60 s) and NMDA 100 microgramsmol litre-1 (30 s), respectively. Thiopental, thiamylal and phenobarbital were present 5 min before, during and 1 min after high K+ or NMDA application. Both thiamylal and thiopental (50-600 microgramsmol litre-1) attenuated the increases in [Ca2+]i produced by high K+ or NMDA in a concentration-dependent manner, while phenobarbital 50-1000 microgramsmol litre- 1 only slightly attenuated the [Ca2+]i increase produced by high K+ at concentrations of more than 200 microgramsmol litre-1 and was ineffective on the [Ca2+]i response produced by NMDA. Although the increases in [Ca2+]i caused by membrane depolarization with high K+ were reduced equally with thiamylal and thiopental, thiamylal was more effective in attenuating the increase in [Ca2+]i produced by NMDA receptor activation than thiopental. We conclude that the depressant effects of barbiturates on both VGCC and NMDA receptors varied between agents. Differential inhibition of both VGCC and NMDA receptors may determine the pharmacological properties of barbiturates and their ability to protect neurones against ischaemia.      

四种静脉麻醉药物对大鼠皮层脑片缺氧缺糖损伤的作用

目的 探讨四种常用静脉麻醉药物对大鼠皮层脑片缺氧缺糖损伤的作用。方法 建立大鼠皮层脑片缺氧缺糖损伤模型,设立对照组、缺氧缺糖损伤组、药物加损伤组,利用2,3,5-三苯基氯化四氮唑(TTC)染色定量比色方法,评价氯胺酮、异丙酚、咪达唑仑和硫喷妥钠对脑片损伤的保护作用。结果 随着缺氧缺糖损伤时间的延长,皮层脑片TTC染色程度明显降低,TTC染色反映的组织损伤百分率与孵育上清液乳酸脱氢酶(LDN)释放比活性呈正相关(r=0.9609,P<0.01)。对于缺氧缺糖损伤所致脑片TTC染色降低,不同浓度氯胺酮均能完全抑制;与损伤组比较。大剂量硫喷妥钠和咪达唑仑(400μmol·L~(-1)和10μmol·L~(-1)A值明显升高(P<0.01或0.05);小剂量异丙酚(1μmol·L~(-1))对脑片TTC染色降低无作用,大剂量(100μmol·L~(-1))加重TTC染色降低(P<0.01)。结论 对于大鼠皮层脑片缺氧缺糖损伤,四种静脉麻醉药物作用效果各不相同:临床麻醉剂量的氯胺酮具有明显保护作用,咪达唑仑和硫喷妥钠在超过临床使用范围的大剂量时有部分保护作用;大剂量异丙酚会加重大鼠皮层脑片的缺氧缺糖损伤。     

异丙酚对新生大鼠海马CA1区兴奋性突触反应的影响

目的：研究异丙酚对新生大鼠海马CAI区兴奋性突触反应的影响。方法：取1周龄Wistar大鼠,快速断头取脑,用振动切片机切取400μm厚的海马脑片,电刺激靠近海马CA1区的Schaffer纤维,用全细胞膜片钳技术记录CA1区锥体细胞的兴奋性突触后电流（excitatory post—synaptic current,EPSC）。循环液中加入不同浓度的异丙酚,观察其对EPSC的影响。然后给与低频刺激（900pulse,3Hz）诱导长时程抑制（10ng—term depression,LTD）,并观察异丙酚对LTD诱导的影响。结果：异丙酚呈剂量依赖性地抑制EPSC,其怍用可被印防己毒素（picrotoxin,pic）阻断;异丙酚可易化由N-甲基-D-门冬氨酸（N—methvl—D—aspartate,NMDA）受体介导的LTD的诱导。结论：异丙酚可影响新生大鼠海马CA1区的兴奋性突触传递和突触可塑性,从而对大鼠的学习和记忆产生影响。     

Modulation of AMPA receptor GluR1 subunit phosphorylation in neurons by the intravenous anaesthetic propofol

Background: The ionotropic glutamate receptor is a potential molecular sitein the central nervous system that general anaesthetics mayinteract with to produce some of their biological actions. Proteinphosphorylation has been well documented to occur in the intracellularC-terminal domain of -amino-3-hydroxy-5-methylisoxazole-4-propionicacid (AMPA) subtype of glutamate receptors, which representsa pivotal mechanism for the post-translational modulation ofAMPA receptor functions. In this study, we investigated a possibleinfluence of an i.v. anaesthetic agent propofol on the phosphorylationof AMPA receptor GluR1 subunits in cultured neurons. Methods: The effect of propofol on phosphorylation of GluR1 subunitsat serine 831 and 845 was assayed in cultured rat striatal andcortical neurons by western blot with phospho- and site-specificantibodies. Results: Propofol consistently elevated phosphorylation of GluR1 subunitsat the C-terminal serine 845 site in both striatal and corticalneurons. The elevation in phosphorylation was concentration-dependentand started at a low concentration (3 µM). This increasein serine 845 phosphorylation was rapid and sustained duringthe entire course of propofol exposure. In contrast to serine845, phosphorylation of GluR1 at serine 831 was not alteredby propofol in striatal and cortical neurons. Total GluR1 abundanceremained unchanged in response to propofol incubation. Conclusions: These data indicate that propofol possesses the ability to upregulateAMPA receptor GluR1 subunit phosphorylation at a specific serine845 site in neurons and provide evidence supporting the AMPAreceptor as a molecular target for general anaesthetics.     

异丙酚对大鼠海马CA1缺血神经元持续钠电流的影响

目的 探讨异丙酚对大鼠海马CA1缺血神经元持续钠电流的影响。方法 酶消化法急性分离SD大鼠海马CA1锥体细胞,通过低氧和无糖法制备神经元缺血模型,全细胞膜片钳技术记录异丙酚对缺血神经元持续钠电流的影响。结果 神经元缺血5 min后持续钠电流显著增强。异丙酚10μmol/L和100μmol/L均能明显抑制缺血引起的持续钠电流增强(与0μmmol/L组比,P<0.01),此作用为异丙酚100μmol/L较10μmol/L儿更强(P<0.05)。结论 异丙酚能够抑制体外脑缺血时海马神经元持续钠电流,这可能是其产生脑保护作用的机制之一。