Christian S. Fahlman

Isoflurane preconditions hippocampal neurons against oxygen-glucose deprivation: role of intracellular Ca2+ and mitogen-activated protein kinase signaling.

Background:Isoflurane preconditions neurons to improve tolerance of subsequent ischemia in both intact animal models and in in vitro preparations. The mechanisms for this protection remain largely undefined. Because isoflurane increases intracellular Ca2+ concentrations and Ca2+ is involved in many processes related to preconditioning, the authors hypothesized that isoflurane preconditions neurons via Ca2+-dependent processes involving the Ca2+- binding protein calmodulin and the mitogen-activated protein kinase–ERK pathway. Methods:The authors used a preconditioning model in which organotypic cultures of rat hippocampus were exposed to 0.5–1.5% isoflurane for a 2-h period 24 h before an ischemia-like injury of oxygen–glucose deprivation. Survival of CA1, CA3, and dentate neurons was assessed 48 later, along with interval measurements of intracellular Ca2+ concentration (fura-2 fluorescence microscopy in CA1 neurons), mitogen-activated protein kinase p42/44, and the survival associated proteins Akt and GSK-3β (in situ immunostaining and Western blots). Results:Preconditioning with 0.5–1.5% isoflurane decreased neuron death in CA1 and CA3 regions of hippocampal slice cultures after oxygen–glucose deprivation. The preconditioning period was associated with an increase in basal intracellular Ca2+ concentration of 7–15%, which involved Ca2+ release from inositol triphosphate–sensitive stores in the endoplasmic reticulum, and transient phosphorylation of mitogen-activated protein kinase p42/44 and the survival-associated proteins Akt and GSK-3β. Preconditioning protection was eliminated by the mitogen-activated extracellular kinase inhibitor U0126, which prevented phosphorylation of p44 during preconditioning, and by calmidazolium, which antagonizes the effects of Ca2+-bound calmodulin. Conclusions:Isoflurane, at clinical concentrations, preconditions neurons in hippocampal slice cultures by mechanisms that apparently involve release of Ca2+ from the endoplasmic reticulum, transient increases in intracellular Ca2+ concentration, the Ca2+ binding protein calmodulin, and phosphorylation of the mitogen-activated protein kinase p42/44.

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 Ca2+ 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.

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°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 &mgr;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 &mgr;m 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 &mgr;m MK-801 in preventing delayed cell death. Modulation of glutamate excitotoxicity may contribute to the protective mechanism.

Redox- and oxidant-mediated regulation of interleukin-10: An anti-inflammatory, antioxidant cytokine?

Reduction-oxidation (redox) state constitutes such a potential signaling mechanism for the regulation of an inflammatory signal associated with oxidative stress. Interleukin (IL)-10 has recently emerged as an anti-inflammatory cytokine with antioxidant properties. Interestingly, redox- and oxidant-mediated pathways positively and/or negatively regulate the expression, distribution, and functional properties of IL-10, thus, allowing the evolution of what is known as an anti-inflammatory redox-oxidant revolving axis. This axis is directly involved in regulating phosphorylation mechanisms, which eventually control gene expression and the biosynthesis of oxidative stress-related cofactors, such as reactive species and inflammatory cytokines. The association between IL-10, an anti-inflammatory antioxidant, with redox- and oxidant-related pathways governing the regulation of inflammatory and closely dependent processes is thereafter discussed.

The inhaled anesthetic, isoflurane, enhances Ca2+-dependent survival signaling in cortical neurons and modulates MAP kinases, apoptosis proteins and transcription factors during hypoxia

We tested whether the protection of hypoxic neurons by the inhaled anesthetic isoflurane is related to the Ca2+-dependent phosphorylation of MAP kinases and anti-apoptotic co-factors. In cultures of mouse cortical neurons we measured changes in the phosphorylation of Ca2+-dependent and Ca2+-independent MAP kinases, transcription factors, and apoptosis regulators after hypoxia or hypoxia combined with isoflurane (1% in gas phase). In hypoxic neurons, isoflurane reduced cell death and TUNEL staining by >80%. Isoflurane released Ca2+ from intracellular stores, increasing [Ca2+]i in oxygenated neurons by approximately 20%. Neuroprotection was associated with a smaller increase in [Ca2+]i in hypoxic neurons and required IP3 receptors and phospholipase C. In hypoxic neurons, isoflurane increased the phosphorylation of the Ca2+-dependent MAP kinases Pyk2 and p42/44 (ERK). The Ca2+-independent MAP kinase p38 pathway showed increased phosphorylation with isoflurane but not with ionomycin, a Ca2+ ionophore. JNK was phosphorylated in hypoxic neurons in the presence of isoflurane, as was the transcription factor c-Jun; JNK inhibition with SP600125 prevented both phosphorylation of c-Jun and neuroprotection. Isoflurane decreased phosphorylation of the pro-apoptotic cofactors Bad and p90RSK and increased Akt phosphorylation. However, with the exception of c-Jun, transcription factors (Elk-1, GSK-3, Forkhead, p90RSK) decreased or remained unchanged. We conclude that isofluranes protection of hypoxic cortical neurons involves signaling that includes changes in intracellular Ca2+ regulation, several MAP kinase pathways and modulation of apoptosis regulators.

Activation of the neuroprotective ERK signaling pathway by fructose-1,6-bisphosphate during hypoxia involves intracellular Ca2+ and phospholipase C

The mechanism of the neuroprotective action of the glycolytic pathway intermediate fructose-1,6-bisphosphate (FBP) may involve activation of a phospholipase-C (PLC) dependent MAP kinase signaling pathway. In this study, we determined whether FBPs capacity to decrease delayed cell death in hippocampal slice cultures is dependent on PLC signaling or activation of the intracellular Ca(2+)-MEK/ERK neuroprotective signaling cascade. FBP (3.5 mM) reduced delayed death from oxygen/glucose deprivation in CA1, CA3 and dentate neurons in slice cultures. The phospholipase-C inhibitor U73122 and the MEK1/2 inhibitor U0126 prevented this protection. In hippocampal and cortical neurons, FBP increased phospho-ERK1/2 (p42/44) immunostaining during hypoxic, but not normoxic conditions. Increased phospho-ERK immunostaining was dependent on PLC and also on MEK 1/2, an upstream regulator of ERK. Further, we found that FBP enhancement of phospho-ERK immunostaining depended on [Ca(2+)](i): PLC inhibition and the IP(3) receptor blocker xestospongin C prevented FBP from increasing [Ca(2+)](i) and increasing phospho-ERK levels. However, while FBP-induced increases in [Ca(2+)](i) were blocked by xestospongin and a PLC inhibitor, [Ca(2+)](i) increases induced by the neuroprotective growth factor BDNF were not prevented. We conclude that during hypoxia FBP initiates a series of neuroprotective signals which include PLC activation, small increases in [Ca(2+)](i), and increased activity of the MEK/ERK signaling pathway.

Inositol 1,4,5-triphosphate receptors and NAD(P)H mediate Ca2+ signaling required for hypoxic preconditioning of hippocampal neurons

Exposure of neurons to a non-lethal hypoxic stress greatly reduces cell death during subsequent severe ischemia (hypoxic preconditioning, HPC). In organotypic cultures of rat hippocampus, we demonstrate that HPC requires inositol triphosphate (IP3) receptor-dependent Ca2+ release from the endoplasmic reticulum (ER) triggered by increased cytosolic NAD(P)H. Ca2+ chelation with intracellular BAPTA, ER Ca2+ store depletion with thapsigargin, IP3 receptor block with xestospongin, and RNA interference against subtype 1 of the IP3 receptor all blunted the moderate increases in [Ca2+](i) (50-100 nM) required for tolerance induction. Increases in [Ca2+](i) during HPC and neuroprotection following HPC were not prevented with NMDA receptor block or by removing Ca2+ from the bathing medium. Increased NAD(P)H fluorescence in CA1 neurons during hypoxia and demonstration that NADH manipulation increases [Ca2+](i) in an IP3R-dependent manner revealed a primary role of cellular redox state in liberation of Ca2+ from the ER. Blockade of IP3Rs and intracellular Ca2+ chelation prevented phosphorylation of known HPC signaling targets, including MAPK p42/44 (ERK), protein kinase B (Akt) and CREB. We conclude that the endoplasmic reticulum, acting via redox/NADH-dependent intracellular Ca2+ store release, is an important mediator of the neuroprotective response to hypoxic stress.

Mild hypothermia, but not propofol, is neuroprotective in organotypic hippocampal cultures

The neuroprotective potency of anesthetics such as propofol compared to mild hypothermia remains undefined. Therefore, we determined whether propofol at two clinically relevant concentrations is as effective as mild hypothermia in preventing delayed neuron death in hippocampal slice cultures (HSC). Survival of neurons was assessed 2 and 3 days after 1 h oxygen and glucose deprivation (OGD) either at 37°C (with or without 10 or 100 &mgr;M propofol) or at an average temperature of 35°C during OGD (mild hypothermia). Cell death in CA1, CA3, and dentate neurons in each slice was measured with propidium iodide fluorescence. Mild hypothermia eliminated death in CA1, CA3, and dentate neurons but propofol protected dentate neurons only at a concentration of 10 &mgr;M; the more ischemia vulnerable CA1 and CA3 neurons were not protected by either 10 &mgr;M or 100 &mgr;M propofol. In slice cultures, the toxicity of 100 &mgr;M N-methyl-d-aspartate (NMDA), 500 &mgr;M glutamate, and 20 &mgr;M &agr;-amino-5-methyl-4-isoxazole propionic acid (AMPA) was not reduced by 100 &mgr;M propofol. Because propofol neuroprotection may involve gamma-aminobutyric acid (GABA)-mediated indirect inhibition of glutamate receptors (GluRs), the effects of propofol on GluR activity (calcium influx induced by GluR agonists) were studied in CA1 neurons in HSC, in isolated CA1 neurons, and in cortical brain slices. Propofol (100 and 200 &mgr;M, approximate burst suppression concentrations) decreased glutamate-mediated [Ca2+]i increases (&Dgr;[Ca2+]i) responses by 25%–35% in isolated CA1 neurons and reduced glutamate and NMDA &Dgr;[Ca2+]i in acute and cultured hippocampal slices by 35%–50%. In both CA1 neurons and cortical slices, blocking GABAA receptors with picrotoxin reduced the inhibition of GluRs substantially. We conclude that mild hypothermia, but not propofol, protects CA1 and CA3 neurons in hippocampal slice cultures subjected to oxygen and glucose deprivation. Propofol was not neuroprotective at concentrations that reduce glutamate and NMDA receptor responses in cortical and hippocampal neurons.

Oxygen sensitivity of NMDA receptors: relationship to NR2 subunit composition and hypoxia tolerance of neonatal neurons

Neonatal rats survive and avoid brain injury during periods of anoxia 25 times longer than adults. We hypothesized that oxygen activates and hypoxia suppresses NMDA receptor (NMDAR) responses in neonatal rat neurons, explaining the innate hypoxia tolerance of these cells. In CA1 neurons isolated from neonatal rat hippocampus (mean postnatal age [P] 5.8 days), hypoxia (PO(2) 10 mm Hg) reduced NMDA receptor-channel open-time percentage and NMDA-induced increase in [Ca(2+)](i) (NMDA DeltaCa(2+)) by 38 and 68% (P<0.01), respectively. In P20 neurons the reductions were not significant. In P3-10 CA1 neurons within intact hippocampal slices, hypoxia reduced NMDA DeltaCa(2+) by 52% (P=0.002) and decreased NMDA-induced death by 45% (P=0.004). Phalloidin, a microtubule stabilizer, prevented hypoxia-induced inhibition of NMDA DeltaCa(2+) in P3-10 neurons. To test whether NMDARs prevalent in neonates (NR1 plus NR2B or NR2D subunits) are inhibited by hypoxia compared with those in mature neurons (NR2A and NR2C), we expressed these receptors in Xenopus oocytes. Compared with responses in 21% O(2), hypoxia (PO(2) 17 mm Hg) reduced currents from neonatal type NR1/NR2D receptors by 25%, increased currents from NR1/NR2C by 18%, and had no effect on NR1/NR2A or NR1/NR2B. Modulation of NMDARs by hypoxia may play an important role in the hypoxia tolerance of the mammalian neonate. In addition, oxygen sensing by NMDARs could play a significant role in postnatal brain development.

Protective effects of TASK-3 (KCNK9) and related 2P K channels during cellular stress

Tandem pore domain (or 2P) K channels form a recently isolated family of channels that are responsible for background K currents in excitable tissues. Previous studies have indicated that 2P K channel activity produces membrane hyperpolarization, which may offer protection from cellular insults. To study the effect of these channels in neuroprotection, we overexpressed pH-sensitive 2P K channels by transfecting the partially transformed C8 cell line with these channels. Tandem pore weak inward rectifier K channel (TWIK)-related acid-sensitive K channel 3 (TASK-3, KCNK9) as well as other pH sensitive 2P K channels (TASK-1 and TASK-2) enhanced cell viability by inhibiting the activation of intracellular apoptosis pathways. To explore the cellular basis for this protection in a more complex cellular environment, we infected cultured hippocampal slices with Sindbis virus constructs containing the coding sequences of these channels. Expression of TASK-3 throughout the hippocampal structure afforded neurons within the dentate and CA1 regions significant protection from an oxygen-glucose deprivation (OGD) injury. Neuroprotection within TASK-3 expressing slices was also enhanced by incubation with isoflurane. These results confirm a protective physiologic capability of TASK-3 and related 2P K channels, and suggest agents that enhance their activity, such as volatile anesthetics may intensify these protective effects.