Roderic G. Eckenhoff

Inhaled Anesthetic Enhancement of Amyloid-β Oligomerization and Cytotoxicity

Background: The majority of surgical patients receive inhaled anesthetics, principally small haloalkanes and haloethers. Long-term cognitive problems occur in the elderly subsequent to anesthesia and surgery, and previous surgery might also be a risk factor for neurodegenerative disorders like Alzheimer and Parkinson disease. The authors hypothesize that inhaled anesthetics contribute to these effects through a durable enhancement of peptide oligomerization. Methods: Light scattering, filtration assays, electron microscopy, fluorescence spectroscopy and size-exclusion chromatography was used to characterize the concentration-dependent effects of halothane, isoflurane, propofol, and ethanol on amyloid &bgr; peptide oligomerization. Pheochromocytoma cells were used to characterize cytotoxicity of amyloid oligomers with and without the above anesthetics. Results: Halothane and isoflurane enhanced amyloid beta oligomerization rates and pheochromocytoma cytotoxicity in vitro through a preference for binding small oligomeric species. Ethanol and propofol inhibited oligomerization at low concentration but enhanced modestly at very high concentration. Neither ethanol nor propofol enhanced amyloid &bgr; toxicity in pheochromocytoma cells. Conclusions: Inhaled anesthetics enhance oligomerization and cytotoxicity of Alzheimer disease-associated peptides. In addition to the possibility of a general mechanism for anesthetic neurotoxicity, these results call for further evaluation of the interaction between neurodegenerative disorders, dementia, and inhalational anesthesia.

Brain and behavior changes in 12-month-old Tg2576 and nontransgenic mice exposed to anesthetics

Inhaled anesthetics have been shown to increase the aggregation of amyloid beta in vitro through the stabilization of intermediate toxic oligomers, which are thought to contribute to neurocognitive dysfunction in Alzheimers disease. Inhaled anesthetics may escalate cognitive dysfunction through enhancement of these intermediate oligomer concentrations. We intermittently exposed 12-month-old Tg2576 transgenic mice and nontransgenic littermates to isoflurane and halothane for 5 days. Cognitive function was measured before and after anesthetic exposures using the Morris Water Maze; amyloid beta plaque burden and caspase-3 mediated apoptosis were quantified by immunohistochemistry. At 12 months of age, anesthetic exposure did not further enhance cognitive decline in the transgenic mice. Immunohistochemistry, however, revealed that the halothane-exposed Tg2576 mice had more amyloidopathy than the isoflurane treated mice or the nonexposed transgenic mice. Isoflurane exposure impaired cognitive function in the nontransgenic mice, implying an alternative pathway for neurodegeneration. These findings indicate that inhaled anesthetics influence cognition and amyloidogenesis, but that the mechanistic relationship remains unclear.

Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently

Depletion of calcium from the neuronal endoplasmic reticulum (ER) induces apoptosis. Isoflurane depletes calcium from sarcoplasmic reticulum (SR) of muscle, an analogue of ER in neurons, while sevoflurane maintains or increases SR calcium. We hypothesized that isoflurane, but not sevoflurane, induces apoptosis by depleting the ER calcium. Rat PC12 pheochromocytoma cells and primary cortical neurons were treated with equipotent doses of isoflurane and sevoflurane. Isoflurane, but not sevoflurane, at equipotent doses induced cell damage determined by both LDH release and MTT reduction assays, dose and time dependently, in both types of cells. Isoflurane at 2.4% for 24 h induced cytotoxicity in both cell types, which was characterized by nuclear condensation and fragmentation and activation of caspases 3 and 9. Isoflurane cytotoxicity was suppressed by dantrolene, a ryanodine receptor antagonist that inhibits abnormal calcium release from the ER. Isoflurane decreased the Bcl-2/Bax ratio by as much as 36% (P < 0.05). However, sevoflurane did not cause neuronal damage by apoptosis nor did it decrease the Bcl-2/Bax ratio. These results suggest that isoflurane and sevoflurane differentially affect the Bcl-2/Bax ratio and cell survival. At equipotent concentrations, isoflurane, but not sevoflurane, induces cytotoxicity in both PC12 cells and primary cortical neurons and decreases the Bcl-2/Bax ratio.

The Common Inhalational Anesthetic Isoflurane Induces Apoptosis via Activation of Inositol 1,4,5-Trisphosphate Receptors

Background:Isoflurane induces cell apoptosis by an unknown mechanism. The authors hypothesized that isoflurane activates inositol 1,4,5-trisphosphate (IP3) receptors on the endoplasmic reticulum (ER) membrane, causing excessive calcium release, triggering apoptosis. Methods:The authors determined isoflurane-induced cytotoxicity by measuring caspase-3 activity, lactate dehydrogenase release, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) reduction, and imaging analysis of cell damage markers (annexin V and propidium iodide staining) in different cell types. The authors used the chicken B lymphocyte with a total knock-out of IP3 receptors, PC12 cells with elevated IP3 receptor activity (transfected with L286V presenilin 1), striatal cells with a knock-in of Q111 Huntingtin, and each cell line’s corresponding wild-type controls. The authors also measured the isoflurane-evoked changes of calcium concentration in cytosol and/or mitochondria in these cells. Results:Isoflurane induced apoptosis concentration- and time-dependently, and sequentially elevated cytosolic and then mitochondrial calcium in the chicken B-lymphocyte wild-type but not the IP3 receptor total knock-out cells. Thapsigargin, a calcium adenosine triphosphatase inhibitor on ER membranes, induced apoptosis and elevations of calcium in cytosol and mitochondria in both chicken B-lymphocyte wild-type and IP3 receptor total knock-out cells. Isoflurane induced significantly more neurotoxicity and greater calcium release from the ER in L286V PC12 and Q111 Huntingtin striatal cells than in their corresponding wild-type controls, both of which were significantly inhibited by the IP3 receptor antagonist xestospongin C. Conclusion:These findings suggest that isoflurane activates the ER membrane IP3 receptor, producing excessive calcium release and triggering apoptosis. Neurons with enhanced IP3 receptor activity, as in certain cases of familial Alzheimer or Huntington disease, may be especially vulnerable to isoflurane cytotoxicity.

Oxygen-dependent lipid peroxidation during lung ischemia

The effect of alveolar oxygen tension on lung lipid peroxidation during lung ischemia was evaluated by using isolated rat lungs perfused with synthetic medium. After a 5-min equilibration period, global ischemia was produced by discontinuing perfusion while ventilation continued with gas mixtures containing 5% CO2 and a fixed oxygen concentration between 0 and 95%. Lipid peroxidation was assessed by measurement of tissue thiobarbituric acid-reactive products and conjugated dienes. Control studies (no ischemia) showed no change in parameters of lipid peroxidation during 1 h of perfusion and ventilation with 20% or 95% O2. With 60 min of ischemia, there was increased lipid peroxidation which varied with oxygen content of the ventilating gas and was markedly inhibited by ventilation with N2. Perfusion with 5-, 8-, 11-, 14-eicosatetraynoic acid indicated that generation of eicosanoids during ischemia accounted for approximately 40-50% of lung lipid peroxide production. Changes of CO2 content of the ventilating gas (to alter tissue pH) or of perfusate glucose concentration had no effect on lipid peroxidation during ischemia, but perfusion at 8% of the normal flow rate prevented lipid peroxidation. Lung dry/wet weight measured after 3 min of reperfusion showed good correlation between lung fluid accumulation and lipid peroxidation. These results indicate that reperfusion is not necessary for lipid peroxidation with ischemic insult of the lung and provide evidence that elevated PO2 during ischemia accelerates the rate of tissue injury.

A Conserved Behavioral State Barrier Impedes Transitions between Anesthetic-Induced Unconsciousness and Wakefulness: Evidence for Neural Inertia

One major unanswered question in neuroscience is how the brain transitions between conscious and unconscious states. General anesthetics offer a controllable means to study these transitions. Induction of anesthesia is commonly attributed to drug-induced global modulation of neuronal function, while emergence from anesthesia has been thought to occur passively, paralleling elimination of the anesthetic from its sites in the central nervous system (CNS). If this were true, then CNS anesthetic concentrations on induction and emergence would be indistinguishable. By generating anesthetic dose-response data in both insects and mammals, we demonstrate that the forward and reverse paths through which anesthetic-induced unconsciousness arises and dissipates are not identical. Instead they exhibit hysteresis that is not fully explained by pharmacokinetics as previously thought. Single gene mutations that affect sleep-wake states are shown to collapse or widen anesthetic hysteresis without obvious confounding effects on volatile anesthetic uptake, distribution, or metabolism. We propose a fundamental and biologically conserved concept of neural inertia, a tendency of the CNS to resist behavioral state transitions between conscious and unconscious states. We demonstrate that such a barrier separates wakeful and anesthetized states for multiple anesthetics in both flies and mice, and argue that it contributes to the hysteresis observed when the brain transitions between conscious and unconscious states.

Embedded cholesterol in the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a cation-selective channel central to both neuronal and muscular processes and is considered the prototype for ligand-gated ion channels, motivating a structural determination effort that spanned several decades [Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J Mol Biol 346:967–989]. Purified nAChR must be reconstituted in a mixture containing cholesterol to function. Proposed modes of interaction between cholesterol and the protein range from specific binding to indirect membrane-mediated mechanisms. However, the underlying cause of nAChR sensitivity to cholesterol remains controversial, in part because the vast majority of functional studies were conducted before a medium resolution structure was reported. We show that the nAChR contains internal sites capable of containing cholesterol, whose occupation stabilizes the protein structure. We detect sites at the protein–lipid interface as conventionally predicted from functional data, as well as deeply buried sites that are not usually considered. Molecular dynamics simulations reveal that occupation of both superficial and deeply buried sites most effectively preserves the experimental structure; the structure collapses in the absence of bound cholesterol. In particular, we find that bound cholesterol directly supports contacts between the agonist-binding domain and the pore that are thought to be essential for activation of the receptor. These results likely apply to those other ion channels within the Cys-loop superfamily that depend on cholesterol, such as the GABA receptor.

Human Alzheimer and inflammation biomarkers after anesthesia and surgery

Background: The prevalence of postoperative cognitive disturbance, coupled with growing in vitro, cell, and animal evidence suggesting anesthetic effects on neurodegeneration, calls for additional study of the interaction between surgical care and Alzheimer neuropathology. The authors studied human cerebrospinal fluid (CSF) biomarkers during surgery. Methods: Eleven patients undergoing idiopathic nasal CSF leak correction were admitted to this Institutional Review Board-approved study. Lumbar subarachnoid catheters were placed before the procedure. Anesthesia was total intravenous propofol or remifentanil or inhalational sevoflurane, depending on provider choice. CSF samples were taken after catheter placement (base), at procedure end (0 h), and then at 6, 24, and 48 h. CSF was analyzed using xMAP Luminex immunoassay (Luminex, Austin, TX). Results: Of the 11 patients (age range, 53 ± 6 yr), 8 were women; 4 received intravenous anesthesia, 6 sevoflurane, and 1 mixed. Procedures lasted 6.4 ± 2 h. Mean CSF amyloid-&bgr;(1–42) remained unchanged, but total-tau and phosphorylated-tau181P increased progressively until at least 48 h. Total-tau, phosphorylated-tau, or amyloid-&bgr;(1–42) concentrations were not different between anesthetic groups. CSF interleukin-10, S100Beta, and tumor necrosis factor &agr; were increased similarly in both anesthetic groups at 24 h, but interleukin-6 was increased more in the inhalational group. Conclusion: These data indicate a robust neuroinflammatory response, including not only the usual markers (interleukin-6, tumor necrosis factor &agr;, interleukin-10), but also S100Beta and tau, markers of injury. The total-tau/amyloid-&bgr;(1–42) ratio increased in a pattern consistent with Alzheimer disease, largely because of an increase in total-tau rather than a decline in amyloid-&bgr;(1–42). The differences in CSF interleukin-6 concentrations suggest that anesthetic management may make a difference in neuroinflammatory response.

Binding of Halothane to Serum Albumin Demonstrated Using Tryptophan Fluorescence

Background : The site of action of general anesthesia remains controversial, but evidence in favor of specific protein target(s) is accumulating. Saturable binding of halothane to bovine serum albumin (BSA) has recently been reported using photoaffinity labeling and fluorine 19 nuclear magnetic resonance spectroscopy. We report a new approach to study anesthetic binding to soluble proteins, based on native tryptophan fluorescence. Methods : Thymol-free halothane and fatty acid-free BSA were equilibrated in gas-tight Hamilton syringes and dispensed into stoppered quartz cuvettes at predetermined dilutions. Steady-state fluorescence spectroscopy was used to study their interaction. Results : Halothane quenched the tryptophan fluorescence of BSA in a concentration-dependent, saturable manner with a dissociation constant = 1.8 ± 0.2 mM and a Hill number = 1.0 ± 0.1. The two optical isomers of halothane bound to BSA with equal affinity. The ability of halothane to quench BSA tryptophan fluorescence was markedly decreased at pH 3.0 (which causes full uncoiling of BSA), with loss of saturable binding. Diethyl ether displaced a portion of halothane from its binding sites. Circular dichroism spectroscopy revealed no significant effect of halothane or diethyl ether on the secondary structure of BSA. Conclusions : The results suggest that halothane binds in hydrophobic domains containing tryptophan in BSA. This approach may prove useful for studying the interaction of volatile anesthetics and proteins and has the advantage that the location of halothane in the protein is identified.

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.