Philip E. Bickler

Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

Abstract: Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as Δ9‐tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA‐induced calcium flux (ΔCa2+NMDA) in rat brain slices. The presence of anandamide reduced ΔCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting ΔCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences ΔCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated ΔCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA‐stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor‐dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

Effects of lsoflurane and Hypothermia on Glutamate Receptor-mediated Calcium Influx in Brain Slices

Background:To understand how volatile anesthetics protect neurons during cerebral ischemia, we studied the effects of isoflurane on cerebral glutamate receptor-mediated calcium influx. Calcium influx via these key excitatory receptors may mediate pain transmission, memory, and the pathophyslologic sequelae of cerebral anoxia or ischemia. Because cerebral protection by hypothermia may involve a decrease in glutamate receptor activity, we also examined the interaction of temperature and isoflurane on glutamate receptor inhibition. Methods:We measured glutamate receptor-mediated changes in cytosolic calcium in 300-µm-thlck rat cortical brain slices. Temperature was varied to 28, 34, 37, or 39°C and isoflurane partial pressure to 0.016-0.019 atm (equivalent to 1.16 minimum alveolar concentration [MAC], adjusted for temperature and age). Brain slices were loaded with fura-2 to permit measurement of cytosolic free calcium. Calcium changes due to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) (50 µM), to ischemia levels of L-glutamate (1.0 mM) or to simulated ischemia (1.0 mM glutamate, 100 µM NaCN, and 3.5 mM iodoacetate) was then measured. Slice lactate dehydrogenase leakage and adenosine triphosphate were measured as indices of cellular integrity. Results: Isoflurane reduced both L-glutamate and NMDA-mediated calcium fluxes by approximately 60%. Neither the activity of the NMDA receptor nor its inhibition by isoflurane was altered by temperature. The rate of calcium influx during ischemia was significantly reduced both by temperature and by isoflurane (P < 0.05). Adenosine triphosphate loss and lactate dehydrogenase leakage were reduced by isoflurane during simulated ischemia by 37% and 73% (P < 0.05), respectively. Conclusions:(1) At 1.16 MAC, isoflurane potently Inhibits glutamate receptors and delays cellular injury induced by simulated ischemia, and (2) hypothermia does not reduce the Intrinsic activity of cortical glutamate receptors but delays calcium accumulation during simulated ischemia. Isoflurane reduces the severity of key pathophyslologic events in an in vitro model of simulated cerebral ischemia.

Causes of calcium accumulation in rat cortical brain slices during hypoxia and ischemia: role of ion channels and membrane damage

To better understand why neurons accumulate calcium during cerebral ischemia, the influence of specific ion channel inhibitors on the rise in cytosolic free calcium ([Ca2+]c) during hypoxia or ischemia was evaluated in rat cerebrocortical brain slices. [Ca2+]c was measured fluorometrically with the dye fura-2 during hypoxia (95% N2/5% CO2 or 100 microM NaCN), simulated ischemia (100 microM NaCN plus 3.5 mM iodoacetate), or 0.5-1.0 mM glutamate. Hypoxia or ischemia increased [Ca+2]c from 100-250 nM to 1,000-2,500 nM within 3-5 min. Greater than 85% of the calcium accumulation was influx from the extracellular medium. The non-competitive N-methyl-D-aspartate (NMDA) inhibitor MK-801 reduced [Ca2+]c accumulation during hypoxia, but antagonism of alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptors or voltage-gated sodium or calcium channels or Na+/Ca2+ exchangers had no effect. During ischemia, combined antagonism of NMDA, AMPA and voltage-gated sodium channels slowed the rate of calcium accumulation, but not concentration at 5 min. Membrane damage, as indicated by leakage of lactate dehydrogenase into superfusate, occurred coincidentally with calcium influx and ATP loss during both hypoxia and ischemia. We conclude that cytosolic calcium changes during hypoxia or ischemia in cortical brain slices are due to multiple mechanisms, are incompletely inhibited by combined ion channel blockade, and are associated with disruption of cell membrane integrity.

Delayed environmental enrichment reverses sevoflurane-induced memory impairment in rats.

Background: Anesthesia given to immature rodents causes cognitive decline, raising the possibility that the same might be true for millions of children undergoing surgical procedures under general anesthesia each year. We tested the hypothesis that anesthesia-induced cognitive decline in rats is treatable. We also tested if anesthesia-induced cognitive decline is aggravated by tissue injury. Methods: Seven-day old rats underwent sevoflurane anesthesia (1 minimum alveolar concentration, 4 h) with or without tail clamping. At 4 weeks, rats were randomized to environmental enrichment or normal housing. At 8 weeks rats underwent neurocognitive testing, which consisted of fear conditioning, spatial reference memory, and water maze-based memory consolidation tests, and interrogated working memory, short-term memory, and early long-term memory. Results: Sevoflurane-treated rats had a greater escape latency when the delay between memory acquisition and memory retrieval was increased from 1 min to 1 h, indicating that short-term memory was impaired. Delayed environmental enrichment reversed the effects of sevoflurane on short-term memory and generally improved many tested aspects of cognitive function, both in sevoflurane-treated and control animals. The performance of tail-clamped rats did not differ from those rats receiving anesthesia alone. Conclusion: Sevoflurane-induced cognitive decline in rats is treatable. Delayed environmental enrichment rescued the sevoflurane-induced impairment in short-term memory. Tissue injury did not worsen the anesthesia-induced memory impairment. These findings may have relevance to neonatal and pediatric anesthesia.

Consensus statement: First International Workshop on Anesthetics and Alzheimer's disease.

In order to review the current status of the potential relationship between anesthesia and Alzheimer’s disease, a group of scientists recently met in Philadelphia for a full day of presentations and discussions. This special article represents a consensus view on the possible link between Alzheimer’s disease and anesthesia and the steps required to test this more definitively.

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°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%, ≈ 0.45 and 1.5 minimum alveolar concentration), pentothal (50 &mgr;m, ≈ 1 minimum alveolar concentration equivalent) and mild hypothermia (34°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.

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.

Effects of Skin Pigmentation on Pulse Oximeter Accuracy at Low Saturation

Background: It is uncertain whether skin pigmentation affects pulse oximeter accuracy at low HbO2 saturation. Methods: The accuracy of finger pulse oximeters during stable, plateau levels of arterial oxygen saturation (Sao2) between 60 and 100% were evaluated in 11 subjects with darkly pigmented skin and in 10 with light skin pigmentation. Oximeters tested were the Nellcor N-595 with the OxiMax-A probe (Nellcor Inc., Pleasanton, CA), the Novametrix 513 (Novametrix Inc., Wallingford, CT), and the Nonin Onyx (Nonin Inc., Plymouth, MN). Semisupine subjects breathed air-nitrogen-carbon dioxide mixtures through a mouthpiece. A computer used end-tidal oxygen and carbon dioxide concentrations determined by mass spectrometry to estimate breath-by-breath Sao2, from which an operator adjusted inspired gas to rapidly achieve 2- to 3-min stable plateaus of desaturation. Comparisons of oxygen saturation measured by pulse oximetry (Spo2) with Sao2 (by Radiometer OSM3) were used in a multivariate model to determine the interrelation between saturation, skin pigmentation, and oximeter bias (Spo2 − Sao2). Results: At 60–70% Sao2, Spo2 (mean of three oximeters) overestimated Sao2 (bias ± SD) by 3.56 ± 2.45% (n = 29) in darkly pigmented subjects, compared with 0.37 ± 3.20% (n = 58) in lightly pigmented subjects (P < 0.0001). The SD of bias was not greater with dark than light skin. The dark-light skin differences at 60–70% Sao2 were 2.35% (Nonin), 3.38% (Novametrix), and 4.30% (Nellcor). Skin pigment-related differences were significant with Nonin below 70% Sao2, with Novametrix below 90%, and with Nellcor at all ranges. Pigment-related bias increased approximately in proportion to desaturation. Conclusions: The three tested pulse oximeters overestimated arterial oxygen saturation during hypoxia in dark-skinned individuals.

Dark Skin Decreases the Accuracy of Pulse Oximeters at Low Oxygen Saturation: The Effects of Oximeter Probe Type and Gender

INTRODUCTION:Pulse oximetry may overestimate arterial oxyhemoglobin saturation (Sao2) at low Sao2 levels in individuals with darkly pigmented skin, but other factors, such as gender and oximeter probe type, remain less studied. METHODS:We studied the relationship between skin pigment and oximeter accuracy in 36 subjects (19 males, 17 females) of a range of skin tones. Clip-on type sensors and adhesive/disposable finger probes for the Masimo Radical, Nellcor N-595, and Nonin 9700 were studied. Semisupine subjects breathed air-nitrogen-CO2 mixtures via a mouthpiece to rapidly achieve 2- to 3-min stable plateaus of Sao2. Comparisons of Sao2 measured by pulse oximetry (Spo2) with Sao2 (by Radiometer OSM-3) were used in a multivariate model to assess the source of errors. RESULTS:The mean bias (Spo2 − Sao2) for the 70%–80% saturation range was 2.61% for the Masimo Radical with clip-on sensor, −1.58% for the Radical with disposable sensor, 2.59% for the Nellcor clip, 3.6% for the Nellcor disposable, −0.60% for the Nonin clip, and 2.43% for the Nonin disposable. Dark skin increased bias at low Sao2; greater bias was seen with adhesive/disposable sensors than with the clip-on types. Up to 10% differences in saturation estimates were found among different instruments in dark-skinned subjects at low Sao2. CONCLUSIONS:Multivariate analysis indicated that Sao2 level, sensor type, skin color, and gender were predictive of errors in Spo2 estimates at low Sao2 levels. The data suggest that clinically important bias should be considered when monitoring patients with saturations below 80%, especially those with darkly pigmented skin; but further study is needed to confirm these observations in the relevant populations.

Efficiency of airway heat and moisture exchangers in anesthetized humans.

The efficiencies of airway heat and moisture exchanging filters in reducing respiratory water losses and increasing airway temperatures during general anesthesia were studied in five tracheally intubated patients given isoflurane, nitrous oxide, and oxygen anesthesia during controlled ventilation. Filters (Humid-Vent Filter, Humid-Vent 1, Pall Conserve, Siemens 150, and Thermo Vent 600) were placed between the y-piece of the anesthesia circle system and the endotracheal tube for 40 min each. Airway temperature, esophageal temperature, and water loss (determined by weighing expired water collected in CaSO4) were measured every 10 min. All of the filters reached near-maximum efficiency in reducing water losses within 10 min. The Humid-Vent Filter and Siemens 150 filters were most efficient, the Pall Conserve and Thermo Vent 600 less efficient. Airway temperature rapidly increased 2°–8°C during each trial. The more efficient the filter in conserving water, the greater the airway temperature. The respiratory heat conserved by these filters represents 5.5%–7.2% of the estimated total metabolic heat production during anesthesia in adults.