Effects of xenon on in vitro and in vivo models of neuronal injury

BACKGROUND: Xenon, the "inert" gaseous anesthetic, is an antagonist at the N-methyl-D-aspartate (NMDA)-type glutamate receptor. Because of the pivotal role that NMDA receptors play in neuronal injury, the authors investigated the efficacy of xenon as a neuroprotectant in both in vitro and in vivo paradigms. METHODS: In a mouse neuronal-glial cell coculture, injury was provoked either by NMDA, glutamate, or oxygen deprivation and assessed by the release of lactate dehydrogenase into the culture medium. Increasing concentrations of either xenon or nitrogen (10-75% of an atmosphere) were coadministered and maintained until injury was assessed. In separate in vivo experiments, rats were administered N-methyl-dl-aspartate and killed 3 h later. Injury was quantified by histologic assessment of neuronal degeneration in the arcuate nucleus of the hypothalamus. RESULTS: Xenon exerted a concentration-dependent protection against neuronal injury provoked by NMDA (IC(50) = 19 +/- 6% atm), glutamate (IC(50) = 28 +/- 8% atm), and oxygen deprivation (IC(50) = 10 +/- 4% atm). Xenon (60% atm) reduced lactate dehydrogenase release to baseline concentrations with oxygen deprivation, whereas xenon (75% atm) reduced lactate dehydrogenase release by 80% with either NMDA- or glutamate-induced injury. In an in vivo brain injury model in rats, xenon exerted a concentration-dependent protective effect (IC(50) = 78 +/- 8% atm) and reduced the injury by 45% at the highest xenon concentration tested (75% atm). CONCLUSIONS: Xenon, when coadministered with the injurious agent, exerts a concentration-dependent neuroprotective effect at concentrations below which anesthesia is produced in rodents. Unlike either nitrous oxide or ketamine (other anesthetics with NMDA antagonist properties), xenon is devoid of both neurotoxicity and clinically significant adverse hemodynamic properties. Studies are proposed to determine whether xenon can be used as a neuroprotectant in certain clinical settings.     

Xenon Acts by Inhibition of Non-N-methyl-d-aspartate Receptor-mediated Glutamatergic Neurotransmission in Caenorhabditis elegans

Background: Electrophysiologic experiments in rodents have found that nitrous oxide and xenon inhibit N-methyl-d-aspartate (NMDA)-type glutamate receptors. These findings led to the hypothesis that xenon and nitrous oxide along with ketamine form a class of anesthetics with the identical mechanism, NMDA receptor antagonism. Here, the authors ask in Caenorhabditis elegans whether xenon, like nitrous oxide, acts by a NMDA receptor-mediated mechanism.

Methods: Xenon:oxygen mixtures were delivered into sealed chambers until the desired concentration was achieved. The effects of xenon on various behaviors were measured on wild-type and mutant C. elegans strains.

Results: With an EC50 of 15-20 vol% depending on behavioral endpoint, xenon altered C. elegans locomotion in a manner indistinguishable from that of mutants in glutamatergic transmission. Xenon reduced the frequency and duration of backward locomotion without altering its speed or other behaviors tested. Mutation of glr-1, encoding a non-NMDA glutamate receptor subunit, abolished the behavioral effects of xenon; however, mutation of nmr-1, which encodes the pore-forming subunit of an NMDA glutamate receptor previously shown to be required for nitrous oxide action, did not significantly alter xenon response. Transformation of the glr-1 mutant with the wild-type glr-1 gene partially restored xenon sensitivity, confirming that glr-1 was necessary for the full action of xenon.     

Bulleyaconitine A Isolated from Aconitum Plant Displays Long-acting Local Anesthetic Properties In Vitro and In Vivo

Background: Bulleyaconitine A (BLA) is an active ingredient of Aconitum bulleyanum plants. BLA has been approved for the treatment of chronic pain and rheumatoid arthritis in China, but its underlying mechanism remains unclear.

Methods: The authors examined (1) the effects of BLA on neuronal voltage-gated Na+ channels in vitro under the whole cell patch clamp configuration and (2) the sensory and motor functions of rat sciatic nerve after single BLA injections in vivo.

Results: BLA at 10 [mu]m did not affect neuronal Na+ currents in clonal GH3 cells when stimulated infrequently to +50 mV. When stimulated at 2 Hz for 1,000 pulses (+50 mV for 4 ms), BLA reduced the peak Na+ currents by more than 90%. This use-dependent reduction of Na+ currents by BLA reversed little after washing. Single injections of BLA (0.2 ml at 0.375 mm) into the rat sciatic notch not only blocked sensory and motor functions of the sciatic nerve but also induced hyperexcitability, followed by sedation, arrhythmia, and respiratory distress. When BLA at 0.375 mm was coinjected with 2% lidocaine (approximately 80 mm) or epinephrine (1:100,000) to reduce drug absorption by the bloodstream, the sensory and motor functions of the sciatic nerve remained fully blocked for approximately 4 h and regressed completely after approximately 7 h, with minimal systemic effects.     

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.     

Ketamine, but Not S (+)-ketamine, Blocks Ischemic Preconditioning in Rabbit Hearts In Vivo

Background: Ketamine blocks KATP channels in isolated cells and abolishes the cardioprotective effect of ischemic preconditioning in vitro. The authors investigated the effects of ketamine and S (+)-ketamine on ischemic preconditioning in the rabbit heart in vivo.

Methods: In 46 [alpha]-chloralose-anesthetized rabbits, left ventricular pressure (tip manometer), cardiac output (ultrasonic flow probe), and myocardial infarct size (triphenyltetrazolium staining) at the end of the experiment were measured. All rabbits were subjected to 30 min of occlusion of a major coronary artery and 2 h of subsequent reperfusion. The control group underwent the ischemia-reperfusion program without preconditioning. Ischemic preconditioning was elicited by 5-min coronary artery occlusion followed by 10 min of reperfusion before the 30 min period of myocardial ischemia (preconditioning group). To test whether ketamine or S (+)-ketamine blocks the preconditioning-induced cardioprotection, each (10 mg kg-1) was administered 5 min before the preconditioning ischemia. To test any effect of ketamine itself, ketamine was also administered without preconditioning at the corresponding time point.

Results: Hemodynamic baseline values were not significantly different between groups [left ventricular pressure, 107 +/- 13 mmHg (mean +/- SD); cardiac output, 183 +/- 28 ml/min]. During coronary artery occlusion, left ventricular pressure was reduced to 83 +/- 14% of baseline and cardiac output to 84 +/- 19%. After 2 h of reperfusion, functional recovery was not significantly different among groups (left ventricular pressure, 77 +/- 19%; cardiac output, 86 +/- 18%). Infarct size was reduced from 45 +/- 16% of the area at risk in controls to 24 +/- 17% in the preconditioning group (P = 0.03). The administration of ketamine had no effect on infarct size in animals without preconditioning (48 +/- 18%), but abolished the cardioprotective effects of ischemic preconditioning (45 +/- 19%, P = 0.03). S (+)-ketamine did not affect ischemic preconditioning (25 +/- 11%, P = 1.0).     

Isoflurane Depresses the Response of Inspiratory Hypoglossal Motoneurons to Serotonin In Vivo

Background: Endogenous serotonin (5-HT) provides important excitatory drive to inspiratory hypoglossal motoneurons (IHMNs). In vitro studies show that activation of postsynaptic 5-HT receptors decreases a leak K+ channel conductance and depolarizes hypoglossal motoneurons (HMNs). In contrast, volatile anesthetics increase this leak K+ channel conductance, which causes neuronal membrane hyperpolarization and depresses HMN excitability. Clinical studies show upper airway obstruction, indicating HMN depression, even at subanesthetic concentrations. The authors hypothesized that if anesthetic activation of leak K+ channels caused neuronal depression in vivo, this effect could be antagonized with serotonin. In this case, the neuronal response to picoejected serotonin would be greater during isoflurane than with no isoflurane.

Methods: Studies were performed in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The authors studied the effect of approximately 0.3 minimum alveolar concentration (MAC) isoflurane on the spontaneous discharge frequency patterns of single IHMNs and on the neuronal response to picoejection of 5-HT.

Results: Normalized data (mean +/- SD, n = 19) confirmed that 0.3 +/- 0.1 MAC isoflurane markedly reduced the spontaneous peak discharge frequency by 48 +/- 19% (P < 0.001) and depressed the slope of the spontaneous discharge patterns. The increase in neuronal frequency in response to 5-HT was reduced by 34 +/- 22% by isoflurane (P < 0.001).     

Amitriptyline versus Bupivacaine in Rat Sciatic Nerve Blockade

Background: Amitriptyline, a tricyclic antidepressant, is frequently used orally for the management of chronic pain. To date there is no report of amitriptyline producing peripheral nerve blockade. The authors therefore investigated the local anesthetic properties of amitriptyline in rats and in vitro.

Methods: Sciatic nerve blockade was performed with 0.2 ml amitriptyline or bupivacaine at selected concentrations, and the motor, proprioceptive, and nociceptive blockade was evaluated. Cultured rat GH3 cells were externally perfused with amitriptyline or bupivacaine, and the drug affinity toward inactivated and resting Na+ channels was assessed under whole-cell voltage clamp conditions. In addition, use-dependent blockade of these drugs at 5 Hz was evaluated.

Results: Complete sciatic nerve blockade for nociception was obtained with amitriptyline for 217 +/- 19 min (5 mm, n = 8, mean +/- SEM) and for 454 +/- 38 min (10 mm, n = 7) versus bupivacaine for 90 +/- 13 min (15.4 mm, n = 6). The time to full recovery of nociception for amitriptyline was 353 +/- 12 min (5 mm) and 656 +/- 27 min (10 mm) versus 155 +/- 9 min for bupivacaine (15.4 mm). Amitriptyline was approximately 4.7-10.6 times more potent than bupivacaine in binding to the resting channels (50% inhibitory concentration [IC50] of 39.8 +/- 2.7 vs. 189.6 +/- 22.3 [mu]m) at -150 mV, and to the inactivated Na+ channels (IC50 of 0.9 +/- 0.1 vs. 9.6 +/- 0.9 [mu]m) at -60 mV. High-frequency stimulation at 3 [mu]m caused an additional approximately 14% blockade for bupivacaine, but approximately 50% for amitriptyline.     

Direct Neurotoxicity of Tetracaine on Growth Cones and Neurites of Growing Neurons In Vitro

Background: Local anesthetics have direct neurotoxicity on neurons. However, precise morphologic changes induced by the direct application of local anesthetics to neurons have not yet been fully understood. Also, despite the fact that local anesthetics are sometimes applied to the sites where peripheral nerves may be regenerating after injury, the effects of local anesthetics on growing or regenerating neurons have never been studied.

Methods: Three different neuronal tissues (dorsal root ganglion, retinal ganglion cell layer, and sympathetic ganglion chain) were isolated from an age-matched chick embryo and cultured for 20 h. Effects of tetracaine were examined microscopically and by a quantitative morphologic assay, growth cone collapse assay.

Results: Tetracaine induced growth cone collapse and neurite destruction. Three neuronal tissues showed significantly different dose-response, both at 60 min and at 24 h after the application of tetracaine (P < 0.01). The ED50 values (mean +/- SD) at 60 min were 1.53 +/- 1.05 mm in dorsal root ganglion, 0.15 +/- 0.05 mm in retinal, and 0.06 +/- 0.02 mm in sympathetic ganglion chain cultures. The ED50 values at 24 h were 0.43 +/- 0.15 mm in dorsal root ganglion, 0.07 +/- 0.03 mm in retinal, and 0.02 +/- 0.01 mm in sympathetic ganglion chain cultures. Concentration of nerve growth factor in the culture media did not influence the ED50 values. The growth cone collapsing effect was partially reversible in dorsal root ganglion and retinal neurons. However, in the sympathetic ganglion culture, no reversibility was observed after exposure to 1 mm tetracaine for 10 or for 60 min. Bupivacaine had similar neurotoxicity to the three types of growing neurons. (The ED50 values at 60 min were 2.32 +/- 0.50 mm in dorsal root ganglion, 0.96 +/- 0.16 mm in retinal, and 0.18 +/- 0.05 mm in sympathetic ganglion chain cultures. The ED50 values at 24 h were 0.34 +/- 0.09 mm in dorsal root ganglion, 0.21 +/- 0.06 mm in retinal, and 0.45 +/- 0.10 mm in sympathetic ganglion chain cultures.)     

Isoflurane and Pentobarbital Reduce AMPA Toxicity In Vivo in the Rat Cerebral Cortex

Background: Isoflurane and pentobarbital can reduce [alpha]-amino-d-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated toxicity in vitro. However, their effect on AMPA toxicity in vivo is not known. The present study was undertaken to evaluate the effects of isoflurane and pentobarbital on the in vivo neurotoxicity produced by AMPA.

Methods: Wistar-Kyoto rats were allocated to one of seven groups (n = 8 per group): isoflurane 1 minimum alveolar concentration, isoflurane electroencephalogram burst suppression (EEG-BS), low-dose pentobarbital, pentobarbital EEG-BS, NBQX, conscious, and sham groups. AMPA 30 nm was injected into the cortex. An equivalent volume of cerebrospinal fluid was injected into the cortex in the sham group. In the NBQX group, 200 nm NBQX was injected into the cortex with the AMPA. In the isoflurane and pentobarbital groups, anesthesia was maintained for a period of 5 h. Animals in the conscious, NBQX, and sham groups were allowed to awaken immediately after the AMPA injection. Injury to the cortex was evaluated 48 h later.

Results: Isoflurane reduced AMPA-induced cortical injury (4.5 +/- 1.9 mm3 and 1.7 +/- 0.8 mm3 in the 1 minimum alveolar concentration and EEG-BS groups, respectively) in comparison to the conscious group (7.2 +/- 0.8 mm3). Pentobarbital reduced cortical injury when administered in EEG-BS doses (2.2 +/- 0.7 mm3) but not when administered in sedative doses (8.6 +/- 0.9 mm3). NBQX reduced AMPA-induced cortical injury (1.2 +/- 0.5 mm3).     

Differential Protective Effects of Volatile Anesthetics against Renal Ischemia-Reperfusion Injury In Vivo

Background: Volatile anesthetics protect against cardiac ischemia-reperfusion injury via adenosine triphosphate-dependent potassium channel activation. The authors questioned whether volatile anesthetics can also protect against renal ischemia-reperfusion injury and, if so, whether cellular adenosine triphosphate-dependent potassium channels, antiinflammatory effects of volatile anesthetics, or both are involved.

Methods: Rats were anesthetized with equipotent doses of volatile anesthetics (desflurane, halothane, isoflurane, or sevoflurane) or injectable anesthetics (pentobarbital or ketamine) and subjected to 45 min of renal ischemia and 3 h of reperfusion during anesthesia.

Results: Rats treated with volatile anesthetics had lower plasma creatinine and reduced renal necrosis 24-72 h after injury compared with rats anesthetized with pentobarbital or ketamine. Twenty-four hours after injury, sevoflurane-, isoflurane-, or halothane-treated rats had creatinine (+/- SD) of 2.3 +/- 0.7 mg/dl (n = 12), 1.8 +/- 0.5 mg/dl (n = 6), and 2.4 +/- 1.2 mg/dl (n = 6), respectively, compared with rats treated with pentobarbital (5.8 +/- 1.2 mg/dl, n = 9) or ketamine (4.6 +/- 1.2 mg/dl, n = 8). Among the volatile anesthetics, desflurane demonstrated the least reduction in plasma creatinine after 24 h (4.1 +/- 0.8 mg/dl, n = 12). Renal cortices from volatile anesthetic-treated rats demonstrated reduced expression of intercellular adhesion molecule 1 protein and messenger RNA as well as messenger RNAs encoding proinflammatory cytokines and chemokines. Volatile anesthetic treatment reduced renal cortex myeloperoxidase activity and reduced nuclear translocation of proinflammatory nuclear factor [kappa]B. Adenosine triphosphate-dependent potassium channels are not involved in sevoflurane-mediated renal protection because glibenclamide did not block renal protection (creatinine: 2.4 +/- 0.4 mg/dl, n = 3).     

Morphine Preconditions Purkinje Cells against Cell Death under In Vitro Simulated Ischemia-Reperfusion Conditions

Background: Morphine pretreatment via activation of [delta]1-opioid receptors induces cardioprotection. In this study, the authors determined whether morphine preconditioning induces ischemic tolerance in neurons.

Methods: Cerebellar brain slices from adult Sprague-Dawley rats were incubated with morphine at 0.1-10 [mu]m in the presence or absence of various antagonists for 30 min. They were then kept in morphine- and antagonist-free buffer for 30 min before they were subjected to simulated ischemia (oxygen-glucose deprivation) for 20 min. After being recovered in oxygenated artificial cerebrospinal fluid for 5 h, they were fixed for morphologic examination to determine the percentage of undamaged Purkinje cells.

Results: The survival rate of Purkinje cells was significantly higher in slices preconditioned with morphine (>= 0.3 [mu]m) before the oxygen-glucose deprivation (57 +/- 4% at 0.3 [mu]m morphine) than that of the oxygen-glucose deprivation alone (39 +/- 3%, P < 0.05). This morphine preconditioning-induced neuroprotection was abolished by naloxone, a non-type-selective opioid receptor antagonist, by naltrindole, a selective [delta]-opioid receptor antagonist, or by 7-benzylidenenaltrexone, a selective [delta]1-opioid receptor antagonist. However, the effects were not blocked by the [mu]-, [kappa]-, or [delta]2-opioid receptor antagonists, [beta]-funaltrexamine, nor-binaltorphimine, or naltriben, respectively. Morphine preconditioning-induced neuroprotection was partially blocked by the selective mitochondrial adenosine triphosphate-sensitive potassium channel antagonist, 5-hydroxydecanoate, or the mitochondrial electron transport inhibitor, myxothiazol. None of the inhibitors used in this study alone affected the simulated ischemia-induced neuronal death.     

Cholinergic Modulation of Sevoflurane Potency in Cortical and Spinal Networks In Vitro

Background: Victims of organophosphate intoxication with cholinergic crisis may have need for sedation and anesthesia, but little is known about how anesthetics work in these patients. Recent studies suggest that cholinergic stimulation impairs [gamma]-aminobutyric acid type A (GABAA) receptor function. Because GABAA receptors are major targets of general anesthetics, the authors investigated interactions between acetylcholine and sevoflurane in spinal and cortical networks.

Methods: Cultured spinal and cortical tissue slices were obtained from embryonic and newborn mice. Drug effects were assessed by extracellular voltage recordings of spontaneous action potential activity.

Results: Sevoflurane caused a concentration-dependent decrease in spontaneous action potential firing in spinal (EC50 = 0.17 +/- 0.02 mm) and cortical (EC50 = 0.29 +/- 0.01 mm) slices. Acetylcholine elevated neuronal excitation in both preparations and diminished the potency of sevoflurane in reducing action potential firing in cortical but not in spinal slices. This brain region-specific decrease in sevoflurane potency was mimicked by the specific GABAA receptor antagonist bicuculline, suggesting that (1) GABAA receptors are major molecular targets for sevoflurane in the cortex but not in the spinal cord and (2) acetylcholine impairs the efficacy of GABAA receptor-mediated inhibition. The latter hypothesis was supported by the finding that acetylcholine reduced the potency of etomidate in depressing cortical and spinal neurons.     

Cyclooxygenase-2 Mediates Ischemic, Anesthetic, and Pharmacologic Preconditioning In Vivo

Background: Cyclooxygenase-2 (COX-2) mediates the late phase of ischemic preconditioning (IPC), but whether this enzyme modulates early IPC, anesthetic-induced preconditioning (APC), or other forms of pharmacologic preconditioning (PPC) is unknown. The authors tested the hypothesis that COX-2 is an essential mediator of IPC, APC, and PPC in vivo.

Methods: Barbiturate-anesthetized dogs (n = 91) were instrumented for measurement of hemodynamics and randomly assigned to receive IPC (four 5-min coronary occlusions interspersed with 5-min reperfusions), APC (1.0 minimum alveolar concentration of isoflurane for 30 min), or PPC (selective mitochondrial KATP channel opener diazoxide, 2.5 mg/kg intravenous) in the presence or absence of pretreatment with oral aspirin (650 mg), the selective COX-2 inhibitor celecoxib (200 mg), or acetaminophen (500 mg) administered 24, 12, and 2 h before experimentation in 12 separate experimental groups. All dogs were subjected to a 60-min coronary artery occlusion followed by 3 h of reperfusion. Myocardial infarct size and coronary collateral blood flow were quantified with triphenyltetrazolium staining and radioactive microspheres, respectively. Myocardial 6-keto-prostaglandin F1[alpha], a stable metabolite of prostacyclin, was measured (enzyme immunoassay) in separate experiments (n = 8) before and after isoflurane administration, in the presence or absence of celecoxib.

Results: No significant differences in baseline hemodynamics or the left ventricular area at risk for infarction were observed between groups. IPC, isoflurane, and diazoxide all decreased myocardial infarct size (9 +/- 1, 12 +/- 2, and 11 +/- 1%, respectively) as compared with control (30 +/- 1%). Celecoxib alone had no effect on infarct size (26 +/- 3%) but abolished IPC (30 +/- 3%), APC (30 +/- 3%), and PPC (26 +/- 1%). Aspirin (24 +/- 3%) and acetaminophen alone (29 +/- 2%) did not alter infarct size or abolish APC-induced protection (18 +/- 1 and 19 +/- 1%, respectively). Isoflurane increased myocardial 6-keto-prostaglandin F1[alpha] to 463 +/- 267% of baseline in the absence but not in the presence (94 +/- 13%) of celecoxib.     

Inhalation Anesthetics Induce Apoptosis in Normal Peripheral Lymphocytes In Vitro

Background : The authors hypothesized that perioperative lymphocytopenia is partially caused by apoptosis of lymphocytes induced by inhalation anesthetics. Therefore, they evaluated whether sevoflurane and isoflurane induce apoptosis of normal peripheral lymphocytes.

Methods : Normal peripheral blood mononuclear cells were exposed to sevoflurane and isoflurane, and the percentages of apoptotic lymphocytes was measured by Annexin V-fluorescein isothiocyanate-7-amino actinomycin D flow cytometry after 24 h of exposure (0.5, 1.0, and 1.5 mm) and after 6, 12, and 24 h of exposure (1.5 mm). The percentages of lymphocytes with caspase 3-like activity were also measured after 24 h of exposure (1.5 mm).

Results : The percentages of apoptotic lymhocytes were increased in a dose-dependent manner (controls: 5.1 +/- 1.4%; sevo-flurane: 7.3 +/- 1.3% [0.5 mm], 9.1 +/- 1.5% [1.0 mm], 12.6 +/- 2.1% [1.5 mm]; isoflurane: 7.5 +/- 1.6% [0.5 mm], 10.5 +/- 1.5% [1.0 mm], 16.3 +/- 2.7% [1.5 mm]) after 24 h of exposure and in a time-dependent manner (controls: 1.2 +/- 0.4% [6 h], 3.4 +/- 0.7% [12 h], 5.6 +/- 1.2% [24 h]; sevoflurane: 1.8 +/- 0.4% [6 h], 6.4 +/- 1.2% [12 h], 11.3 +/- 2.2% [24 h]; isoflurane: 2.6 +/- 0.5% [6 h], 8.8 +/- 1.5% [12 h],16.0 +/- 1.9% [24 h]) at the concentration of 1.5 mm. The percentages of lymphocytes with caspase 3-like activity were increased (controls: 10.0 +/- 1.1%; sevoflurane: 13.8 +/- 1.2%; isoflurane: 17.0 +/- 1.3%).     

Mechanisms of Desflurane-induced Preconditioning in Isolated Human Right Atria In Vitro

Background: The authors examined the role of adenosine triphosphate-sensitive potassium (KATP) channels, adenosine A1 receptor, and [alpha] and [beta] adrenoceptors in desflurane-induced preconditioning in human myocardium, in vitro.

Methods: The authors recorded isometric contraction of human right atrial trabeculae suspended in oxygenated Tyrode's solution (34[degrees]C; stimulation frequency, 1 Hz). Before a 30-min anoxic period, 3, 6, and 9% desflurane was administered during 15 min. Desflurane, 6%, was also administered in the presence of 10 [mu]m glibenclamide, a KATP channels antagonist; 10 [mu]m HMR 1098, a sarcolemmal KATP channel antagonist; 800 [mu]m 5-hydroxy-decanoate (5-HD), a mitochondrial KATP channel antagonist; 1 [mu]m phentolamine, an [alpha]-adrenoceptor antagonist; 1 [mu]m propranolol, a [beta]-adrenoceptor antagonist; and 100 nm 8-cyclopentyl-1,3-dipropylxanthine (DPX), the adenosine A1 receptor antagonist. Developed force at the end of a 60-min reoxygenation period was compared (mean +/- SD).

Results: Desflurane at 3% (95 +/- 13% of baseline), 6% (86 +/- 6% of baseline), and 9% (82 +/- 6% of baseline) enhanced the recovery of force after 60 min of reoxygenation as compared with the control group (50 +/- 11% of baseline). Glibenclamide (60 +/- 12% of baseline), 5-HD (57 +/- 21% of baseline), DPX (63 +/- 19% of baseline), phentolamine (56 +/- 20% of baseline), and propranolol (63 +/- 13% of baseline) abolished desflurane-induced preconditioning. In contrast, HMR 1098 (85 +/- 12% of baseline) did not modify desflurane-induced preconditioning.     

Activation of A3 Adenosine Receptors Attenuates Lung Injury after In Vivo Reperfusion

Background: A3 adenosine receptor (AR) activation worsens or protects against renal and cardiac ischemia-reperfusion (IR) injury, respectively. The aims of the current study were to examine in an in vivo model the effect of A3AR activation on IR lung injury and investigate the mechanism by which it exerts its effect.

Methods: The arterial branch of the left lower lung lobe in intact-chest, spontaneously breathing cats was occluded for 2 h and reperfused for 3 h (IR group). Animals were treated with the selective A3 receptor agonist IB-MECA (300 [mu]g/kg intravenously) given 15 min before ischemia or with IB-MECA as described, with pretreatment 15 min earlier with the selective A3AR antagonist MRS-1191, the nonsulfonylurea adenosine triphosphate-sensitive potassium channel-blocking agent U-37883A, or the nitric oxide synthase inhibitor Nw-nitro-l-arginine benzyl ester.

Results: IB-MECA markedly (P < 0.01) reduced the percentage of injured alveoli (IR, 48 +/- 4%; IB-MECA, 18 +/- 2%), wet:dry weight ratio (IR, 8.2 +/- 0.4; IB-MECA, 4 +/- 2), and myeloperoxidase activity (IR, 0.52 +/- 0.06 U/g; IB-MECA, 0.17 +/- 0.04 U/g). This protective effect was completely blocked by pretreatment with the selective A3AR antagonist MRS-1191 and the adenosine triphosphate-sensitive potassium channel blocking agent U-37883A but not the nitric oxide synthase inhibitor Nw-nitro-l-arginine benzyl ester.     

Isoflurane Preconditions Myocardium against Infarction via Release of Free Radicals

Background: Isoflurane exerts cardioprotective effects that mimic the ischemic preconditioning phenomenon. Generation of free radicals is implicated in ischemic preconditioning. The authors investigated whether isoflurane-induced preconditioning may involve release of free radicals.

Methods: Sixty-one [alpha]-chloralose-anesthetized rabbits were instrumented for measurement of left ventricular (LV) pressure (tip-manometer), cardiac output (ultrasonic flowprobe), and myocardial infarct size (triphenyltetrazolium staining). All rabbits were subjected to 30 min of occlusion of a major coronary artery and 2 h of subsequent reperfusion. Rabbits of all six groups underwent a treatment period consisting of either no intervention for 35 min (control group, n = 11) or 15 min of isoflurane inhalation (1 minimum alveolar concentration end-tidal concentration) followed by a 10-min washout period (isoflurane group, n = 12). Four additional groups received the radical scavenger N-(2-mercaptoproprionyl)glycine (MPG; 1 mg [middle dot] kg-1 [middle dot] min-1) or Mn(III)tetrakis(4-benzoic acid)porphyrine chloride (MnTBAP; 100 [mu]g [middle dot] kg-1 [middle dot] min-1) during the treatment period with (isoflurane + MPG; n = 11; isoflurane + MnTBAP, n = 9) or without isoflurane inhalation (MPG, n = 11; MnTBAP, n = 7).

Results: Hemodynamic baseline values were not significantly different between groups (LV pressure, 97 +/- 17 mmHg [mean +/- SD]; cardiac output, 228 +/- 61 ml/min). During coronary artery occlusion, LV pressure was reduced to 91 +/- 17% of baseline and cardiac output to 94 +/- 21%. After 2 h of reperfusion, recovery of LV pressure and cardiac output was not significantly different between groups (LV pressure, 83 +/- 20%; cardiac output, 86 +/- 23% of baseline). Infarct size was reduced from 49 +/- 17% of the area at risk in controls to 29 +/- 19% in the isoflurane group (P = 0.04). MPG and MnTBAP themselves had no effect on infarct size (MPG, 50 +/- 14%; MnTBAP, 56 +/- 15%), but both abolished the preconditioning effect of isoflurane (isoflurane + MPG, 50 +/- 24%, P = 0.02; isoflurane + MnTBAP, 55 +/- 10%, P = 0.001).     

Oxygen Attenuates Atelectasis-induced Injury in the In Vivo Rat Lung

Background: Atelectasis results in impaired compliance and gas exchange and, in extreme cases, increased microvascular permeability, pulmonary hypertension, and right ventricular dysfunction. It is not known whether such atelectasis-induced lung injury is due to the direct mechanical effects of lung volume reduction and alveolar collapse or due to the associated regional lung hypoxia. The authors hypothesized that addition of supplemental oxygen to an atelectasis-prone ventilation strategy would attenuate the pulmonary vascular effects and reduce the local levels of vasoconstrictor eicosanoids.

Methods: In series 1, anesthetized, atelectasis-prone mechanically ventilated rats were randomly assigned to one of six groups based on the inspired oxygen concentration and ventilated without recruitment. Series 2 was performed to determine the cardiac and pulmonary vascular effects of 21% versus 100% inspired oxygen. In series 3, computed tomography scans were performed after ventilation with a recruitment strategy (21% O2) or no recruitment strategy (21% O2 or 100% O2). In series 4, functional residual capacity was measured in animals where the gas was 21% or 100% O2.

Results: The partial pressure of arterial oxygen increased with increasing inspired oxygen, but the alveolar-arterial oxygenation gradient was also greater with higher inspired oxygen. Ventilation with 21% O2 (but not with 100% O2) was associated with progressive pulmonary vascular impedance and increased pulmonary vascular permeability. Prostaglandin F2[alpha] was increased by mechanical ventilation, especially without supplemental oxygen. Computed tomography scans demonstrated no atelectasis in recruited lungs, and atelectasis in nonrecruited lungs that was greater with supplemental oxygen. Increased atelectasis with 100% O2 (vs. 21% O2) was demonstrated by measurement of functional residual capacity.     

In Vitro and In Vivo Effects of the Phosphodiesterase-III Inhibitor Enoximone on Malignant Hyperthermia-susceptible Swine

Background: In human skeletal muscles, the phosphodiesterase-III inhibitor enoximone induces in vitro contracture development, and it has been suggested that enoximone could trigger malignant hyperthermia (MH). In this study, the in vitro and in vivo effects of enoximone in MH-normal (MHN) and MH-susceptible (MHS) swine were investigated.

Methods: Malignant hyperthermia trigger-free general anesthesia was performed in MHS and MHN swine. Skeletal muscle specimens were excised for an in vitro contracture test with 0.6 mm enoximone. Thereafter, MHS and MHN swine were exposed to cumulative administration of 0.5, 1, 2, 4, 8, 16, and 32 mg/kg enoximone intravenously. Clinical occurrence of MH was defined by a Pco2 greater than 70 mmHg, a pH less than 7.20, and an increase in body temperature of more than 2.0[degrees]C.

Results: Enoximone induced marked contractures in all MHS muscle specimens in vitro. In contrast, only small or no contracture development was observed in MHN muscle specimens, without an overlap in contractures between MHS and MHN muscles. However, in vivo, no clinical differences were found between MHS and MHN swine following cumulative enoximone doses. According to the defined criteria, none of the swine developed MH during the experiment. Furthermore, high enoximone doses induced progressive circulatory insufficiency, and after receiving 32 mg/kg enoximone, all animals died of cardiovascular failure.     

The Quaternary Lidocaine Derivative, QX-314, Produces Long-lasting Local Anesthesia in Animal Models In Vivo

Background: QX-314 is a quaternary lidocaine derivative considered to be devoid of clinically useful local anesthetic activity. However, several reports document that extracellular QX-314 application affects action potentials. Hence, the authors tested the hypothesis that QX-314 could produce local anesthesia in animal models in vivo.

Methods: The authors tested QX-314 (10, 30, and 70 mm) in three standard in vivo local anesthetic animal models, using a randomized, blinded experimental design with negative (placebo) and positive (70 mm lidocaine) controls. The guinea pig intradermal wheal assay (n = 29) was used to test for peripheral inhibition of the cutaneous trunci muscle reflex, the mouse tail-flick test (n = 30) was used to test for sensory blockade, and the mouse sciatic nerve blockade model (n = 45) was used to test for motor blockade.

Results: In all three animal models, QX-314 concentration-dependently and reversibly produced local anesthesia of long duration, at concentrations equivalent to those clinically relevant for lidocaine. In the guinea pig intradermal wheal assay, QX-314 produced peripheral nociceptive blockade up to 6 times longer than lidocaine (650 +/- 171 vs. 100 +/- 24 min [mean +/- SD]; n = 6 per group; P < 0.0001). In the mouse tail-flick test, QX-314 produced sensory blockade up to 10 times longer than lidocaine (540 +/- 134 vs. 50 +/- 11 min; n = 6 per group; P < 0.0001). Finally, in the mouse sciatic nerve model, QX-314 produced motor blockade up to 12 times longer compared with lidocaine (282 +/- 113 vs. 23 +/- 10 min; n = 9 or 10 per group; P < 0.0001). The onset of QX-314-mediated blockade was consistently slower compared with lidocaine. Animals injected with saline exhibited no local anesthetic effects in any of the three models.