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1.  [No title available] 
PMCID: PMC3947586  PMID: 24322588
2.  [No title available] 
PMCID: PMC4102494  PMID: 24448089
3.  Endothelial Barrier Protection by Local Anesthetics: Ropivacaine and Lidocaine Block Tumor Necrosis Factor-α–induced Endothelial Cell Src Activation 
Anesthesiology  2014;120(6):1414-1428.
Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase–Akt–nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability.
Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed.
Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10−10 M for ropivacaine; IC50 = 5.864 × 10−10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10−10 M for ropivacaine; IC50 = 6.377 × 10−10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated.
Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory “side-effect” of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease.
PMCID: PMC4284094  PMID: 24525631
4.  Immobilization with Atrophy Induces Increased Expression of Neuronal Nicotinic α7 Acetylcholine Receptors in Muscle Contributing to Neurotransmission 
Anesthesiology  2014;120(1):76-85.
Mature acetylcholine receptor (AChR) isoform normally mediates muscle contraction. The hypothesis that α7AChRs upregulate during immobilization and contribute to neurotransmission was tested pharmacologically using specific blockers to mature (waglerin-1), immature (αA-OIVA), and α7AChRs methyllycaconitine, and non-specific muscle AChR antagonist, α-bungarotoxin.
Mice were immobilized; contralateral limb was control. Fourteen days later, anesthetized mice were mechanically ventilated. Nerve-stimulated tibialis muscle contractions on both sides were recorded, and blockers enumerated above sequentially administered via jugular vein. Data are mean ± S.E.
Immobilization (N=7) induced tibialis muscle atrophy (40.6 ± 2.8 vs 52.1 ± 2.0 mg, p<0.01) and decrease of twitch tension (34.8 ± 1.1 vs. 42.9 ± 1.5 g, p< 0.01). Waglerin-1 (0.3 ± 0.05 μg/g) significantly (p=0.001, N=9) depressed twitch tension on contralateral (≥ 97%) versus immobilized side (~45%). Additional waglerin-1 (total dose 1.06 ± 0.12 μg/g or ~15.0 X ED50 in normals) could not depress twitch ≥ 80% on immobilized side. Immature AChR blocker, αA-OIVA (17.0 ± 0.25 μg/g) did not change tension bilaterally. Administration of α-bungarotoxin (N=4) or methyllycaconitine (N=3) caused ≥ 96% suppression of the remaining twitch tension on immobilized side. Methyllycaconitine, administered first (N=3), caused equipotent inhibition by waglerin-1 on both sides. Protein expression of α7AChRs was significantly (N=3, p<0.01) increased on the immobilized side.
Ineffectiveness of waglerin-1 suggests the twitch tension during immobilization is maintained by receptors other than mature AChRs. Since αA-OIVA caused no changes, immature AChRs contribute minimally to neurotransmission. During immobilization ~20% of twitch tension is maintained by upregulation of α-bungarotoxin- and methyllycaconitine-sensitive α7AChRs.
PMCID: PMC3910258  PMID: 24126263
5.  Automated Near Real-Time Clinical Performance Feedback for Anesthesiology Residents: One Piece of the Milestones Puzzle 
Anesthesiology  2014;120(1):172-184.
Anesthesiology residencies are developing trainee assessment tools to evaluate 25 milestones that map to the 6 core competencies. The effort will be facilitated by development of automated methods to capture, assess, and report trainee performance to program directors, the Accreditation Council for Graduate Medical Education and the trainees themselves.
We leveraged a perioperative information management system to develop an automated, near-real-time performance capture and feedback tool that provides objective data on clinical performance and requires minimal administrative effort. Prior to development, we surveyed trainees about satisfaction with clinical performance feedback and about preferences for future feedback.
Resident performance on 24,154 completed cases has been incorporated into our automated dashboard, and trainees now have access to their own performance data. Eighty percent (48 of 60) of our residents responded to the feedback survey. Overall, residents ‘agreed/strongly agreed’ that they desire frequent updates on their clinical performance on defined quality metrics and that they desired to see how they compared to the residency as a whole. Prior to deployment of the new tool, they ‘disagreed’ that they were receiving feedback in a timely manner. Survey results were used to guide the format of the feedback tool that has been implemented.
We demonstrate the implementation of a system that provides near real-time feedback concerning resident performance on an extensible series of quality metrics, and which is responsive to requests arising from resident feedback about desired reporting mechanisms.
PMCID: PMC3893706  PMID: 24398735
6.  Facilitation of Resident Scholarly Activity: Strategy and Outcome Analyses Using Historical Resident Cohorts and a Rank-to-Match Population 
Anesthesiology  2014;120(1):111-119.
Facilitation of residents’ scholarly activities is indispensable to the future of medical specialties. Research education initiatives and their outcomes, however, have rarely been reported.
Since Academic Year (AY) 2006, research education initiatives, including research lectures, research problem based learning discussions, and an elective research rotation under a new research director’s supervision, have been used. The effectiveness of the initiatives was evaluated by comparing the number of residents and faculty mentors involved in residents’ research activity (Pre-initiative [2003 to 2006] vs. Post-initiative [2007 to 2011]). The residents’ current post-graduation practices were also compared. To minimize potential historical confounding factors, peer reviewed publications based on work performed during residency written by residents who graduated from the program in AY2009 to AY2011 were further compared with those of rank-to-match residents, who were on the residency ranking list during the same AYs and could have been matched with our program had they ranked it high enough on their list.
The Post-initiative group showed greater resident research involvement compared to the Pre-initiative group (89.2% [58 in 65 residents] vs. 64.8% [35 in 54], p=0.0013) and greater faculty involvement (23.9% [161 in 673 faculty/year] vs. 9.2% [55 in 595], p<0.0001). Choice of academic practice did not increase (50.8% [Post] vs. 40.7% [Pre], p=0.36). Graduated residents (n=38) published more often than the rank-to-match residents (n=220) (55.3% [21 residents] vs. 13.2% [29], p<0.0001, odds ratio 8.1 with 95% confidence interval of 3.9 to 17.2).
Research education initiatives increased residents’ research involvement.
PMCID: PMC3905449  PMID: 24212198
7.  Despite Differences in Cytosolic Calcium Regulation, Lidocaine Toxicity Is Similar in Adult and Neonatal Rat Dorsal Root Ganglia in Vitro 
Anesthesiology  2014;120(1):50-61.
Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action for local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, we examined whether there were differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons.
DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine.
The mean KCl-induced calcium transient was greater in P7 neurons (p < 0.05), and lidocaine significantly inhibited KCl-induced calcium responses in both ages (p < 0.05). Frequency distribution histograms of KCl-evoked calcium increases were more heterogeneous in P7 than in adult neurons. With lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages.
Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.
PMCID: PMC3947281  PMID: 23851347
8.  Predictors of Survival from Perioperative Cardiopulmonary Arrests: A Retrospective Analysis of 2,524 Events from the National Registry of Cardiopulmonary Resuscitation 
Anesthesiology  2013;119(6):10.1097/ALN.0b013e318289bafe.
Perioperative cardio-pulmonary arrests are uncommon and little is known about rates and predictors of in-hospital survival.
Using the Get-With-The-Guidelines – Resuscitation national cardiopulmonary resuscitation registry, we identified all patients aged 18 years or older who experienced an index, pulseless cardiac arrest in the operating room or within 24 hours postoperatively. The primary outcome was survival to hospital discharge, and the secondary outcome was neurologically intact recovery among survivors. Multivariable logistic regression models using generalized estimating equation models were used to identify independent predictors of survival and neurologically intact survival.
There were 2,524 perioperative cardiopulmonary arrests identified from 234 hospitals. The overall rate of survival to discharge was 31.7% (799/2,524), including 41.8% (254/608) for ventricular tachycardia and ventricular fibrillation, 30.5% (296/972) for asystole, and 26.4% (249/944) for pulseless electrical activity. Ventricular fibrillation and pulseless ventricular tachycardia were independently associated with improved survival. Asystolic arrests occurring in the operating room and post-anesthesia care unit were associated with improved survival when compared to other perioperative locations. Among patients with neurological status assessment at discharge, the rate of neurologically intact survival was 64.0% (473/739). Pre-arrest neurological status at admission, patient age, inadequate natural airway, pre-arrest ventilatory support, duration of event and event location were significant predictors of neurological status at discharge.
Among patients with a perioperative cardiac arrest, 1 in 3 survived to hospital discharge, and good neurological outcome was noted in 2 out of 3 survivors.
PMCID: PMC3797152  PMID: 23838723
9.  Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice 
Anesthesiology  2013;119(6):10.1097/ALN.0b013e3182a950da.
Isoflurane releases renal tubular transforming growth factor-beta 1 (TGF-β1) and protects against ischemic acute kidney injury (AKI). Recent studies suggest that TGF-β1 can induce a cytoprotective cytokine interleukin (IL)-11. Here, we tested the hypothesis that isoflurane protects against ischemic AKI by direct induction of renal tubular IL-11 synthesis.
Human kidney proximal tubule (HK-2) cells were treated with 1.25-2.5% isoflurane or carrier gas (room air+5% carbon dioxide) for 0-16 h. We also anesthetized C57BL/6 mice with 1.2% isoflurane or with equi-anesthetic dose of pentobarbital for 4 h. In addition, we subjected IL-11 receptor (IL-11R) wild type, IL-11R deficient or IL-11 neutralized mice to 30-min renal ischemia followed by reperfusion under 4 h of pentobarbital or isoflurane (1.2%) anesthesia.
Isoflurane increased IL-11 synthesis in human (~300-500% increase, N = 6) and mouse (23 ± 4 (mean ± SD) fold over carrier gas group, N = 4) proximal tubule cells that were attenuated by a TGF-β1 neutralizing antibody. Mice anesthetized with isoflurane showed significantly increased kidney IL-11 messenger RNA (13.8 ± 2 fold over carrier gas group, N = 4) and protein (31 ± 9 vs. 18±2 pg/mg protein or ~80% increase, N = 4) expression compared to pentobarbital anesthetized mice and this increase was also attenuated by a TGF-β1 neutralizing antibody. Furthermore, isoflurane-mediated renal protection in IL-11R wild-type mice were absent in IL-11R deficient mice or in IL-11R wild-type mice treated with IL-11 neutralizing antibody (N = 4-6).
Our studies suggest that isoflurane induces renal tubular IL-11 via TGF-β1 signaling to protect against ischemic AKI.
PMCID: PMC3867581  PMID: 24037316
10.  Survey Criteria for Fibromyalgia Independently Predict Increased Postoperative Opioid Consumption after Lower Extremity Joint Arthroplasty: A Prospective, Observational Cohort Study 
Anesthesiology  2013;119(6):10.1097/ALN.0b013e3182a8eb1f.
Variance in pain following total knee and hip arthroplasty may be due to a number of procedural and peripheral factors but also, in some individuals, to aberrant central pain processing as is described in conditions like fibromyalgia. To test this hypothesis, we conducted a prospective, observational cohort study of patients undergoing lower extremity joint arthroplasty.
519 patients were preoperatively phenotyped using validated self-reported pain questionnaires, psychological measures, and health information. In addition to assessing factors previously found to be associated with poor outcomes in arthroplasty, participants also completed the American College of Rheumatology survey criteria for fibromyalgia. Previous studies have suggested that rather than being “present” or “absent,” features of fibromyalgia as measured by this instrument, occur over a wide continuum. Postoperative pain control was assessed by total postoperative opioid consumption.
Preoperatively, patients with higher fibromyalgia survey scores were younger, more likely to be female, taking more opioids, reported higher pain severity, and had a more negative psychological profile. In the multivariate analysis, the fibromyalgia survey score, younger age, preoperative opioid use, knee (vs. hip), pain severity at baseline, and the anesthetic technique were all predictive of increased postoperative opioid consumption.
Using the survey criteria for fibromyalgia distinct phenotypic differences were found, and the measure was independently predictive of opioid consumption. This self-report measure may provide an additional simple means of predicting postoperative pain outcomes and analgesic requirements. Future studies are needed to determine whether tailored therapies can improve postoperative pain control in this population.
PMCID: PMC3867739  PMID: 24343289
11.  Reconfiguration of Network Hub Structure after Propofol-Induced Unconsciousness 
Anesthesiology  2013;119(6):10.1097/ALN.0b013e3182a8ec8c.
General anesthesia induces unconsciousness along with functional changes in brain networks. Considering the essential role of hub structures for efficient information transmission, we hypothesized that anesthetics have an effect on the hub structure of functional brain networks.
Graph theoretical network analysis was carried out to study the network properties of 21-channel electroencephalogram data from ten human volunteers anesthetized on two occasions. The functional brain network was defined by phase lag index, a coherence measure, for three states: wakefulness, loss of consciousness induced by the anesthetic propofol, and recovery of consciousness. The hub nodes were determined by the largest centralities. The correlation between the altered hub organization and the phase relationship between electroencephalographic channels was investigated.
Topology rather than connection strength of functional networks correlated with states of consciousness. The average path length, clustering coefficient and modularity significantly increased after administration of propofol, which disrupted long range connections. In particular, the strength of hub nodes significantly decreased. The primary hub location shifted from the parietal to frontal region, in association with propofol-induced unconsciousness. The phase lead of frontal to parietal regions in the alpha frequency band (8-13 Hz) observed during wakefulness reversed direction after propofol and returned during recovery.
Propofol reconfigures network hub structure in the brain and reverses the phase relationship between frontal and parietal regions. Changes in network topology are more closely associated with states of consciousness than connectivity and may be the primary mechanism for the observed loss of frontal-to-parietal feedback during general anesthesia.
PMCID: PMC3873632  PMID: 24013572
12.  Perioperative Organ Injury 
Anesthesiology  2013;119(6):1474-1489.
In spite of the fact that a surgical procedure may have been performed for the appropriate indication and in a technically perfect manner, patients are threatened by perioperative organ injury. For example, stroke, myocardial infarction, acute respiratory distress syndrome, acute kidney injury, or acute gut injury are among the most common causes for morbidity and mortality in surgical patients. In the present review, we discuss the pathogenesis of perioperative organ injury, and provide select examples for novel treatment concepts that have emerged over the past decade. Indeed, we believe that research to provide mechanistic insight into acute organ injury and to identify novel therapeutic approaches for the prevention or treatment of perioperative organ injury represents the most important opportunity to improve outcomes of anesthesia and surgery.
PMCID: PMC3929362  PMID: 24126264
13.  Positive End Expiratory Pressure Increments during Anesthesia in Normal Lung Result in Hysteresis and Greater Numbers of Smaller Aerated Airspaces 
Anesthesiology  2013;119(6):1402-1409.
While it is recognized that pulmonary hysteresis can influence the effects of positive end-expiratory pressure (PEEP), the extent to which expansion of previously opened (vs. newly opening) peripheral airspaces contribute to increased lung volume is not known.
Following a recruitment maneuver, rats were ventilated with constant tidal volumes and imaged during ascending and descending ramps of PEEP.
We estimated peripheral airspace dimensions by measuring the apparent diffusion coefficient (ADC) of 3He in 10 rats. In a separate group (n = 5) undergoing a similar protocol, we used computerized tomography to quantify lung volume. Hysteresis was confirmed by larger end-inspiratory lung volume (mean ± SD; all PEEP levels included): 8.4 ± 2.8 versus 6.8 ± 2.0 mL (P < 0.001) and dynamic compliance: 0.52 ± 0.12 versus 0.42 ± 0.09 mL/cmH2O (P < 0.001) during descending versus ascending PEEP ramps. ADC increased with PEEP but it was smaller during the descending versus ascending ramps for corresponding levels of PEEP: 0.168 ± 0.019 versus 0.183 ± 0.019 cm2/s (P < 0.001). ADC was smaller in the posterior versus anterior lung regions, but the effect of PEEP and hysteresis on ADC was greater in the posterior regions.
Our results suggest that in healthy lungs, larger lung volumes due to hysteresis are associated with smaller individual airspaces. This may be explained by opening of previously non-aerated peripheral airspaces, rather than expansion of those already aerated. Setting PEEP on a descending ramp may minimize distension of individual airspaces.
PMCID: PMC3987989  PMID: 24025616
14.  Consciousness and the 21st Century Operating Room 
Anesthesiology  2013;119(5):10.1097/ALN.0b013e3182a7cad1.
PMCID: PMC3823752  PMID: 23962968
15.  Assessment of Homology Templates and an Anesthetic Binding Site within the γ-Aminobutyric Acid Receptor 
Anesthesiology  2013;119(5):10.1097/ALN.0b013e31829e47e3.
Anesthetics mediate portions of their activity via modulation of the γ-aminobutyric acid receptor (GABAaR). While its molecular structure remains unknown, significant progress has been made towards understanding its interactions with anesthetics via molecular modeling.
The structure of the torpedo acetylcholine receptor (nAChRα), the structures of the α4 and β2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50’s.
Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between alpha and beta subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50.
Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed correlation of ligand docking scores with experimentally measured GABAaR potentiation.
PMCID: PMC3823821  PMID: 23770602
16.  Substance P saporin for bone cancer pain in dogs: Can man’s best friend solve the lost in translation problem in analgesic development? 
Anesthesiology  2013;119(5):10.1097/ALN.0b013e3182a951a2.
PMCID: PMC3827733  PMID: 24195943
17.  Intrathecal substance-p saporin in the dog: Efficacy in bone cancer pain 
Anesthesiology  2013;119(5):10.1097/ALN.0b013e3182a95188.
Substance-p saporin (SP-SAP), a chemical conjugate of substance-p and a recombinant version of the ribosome-inactivating protein, saporin, when administered intrathecally, acts as a targeted neurotoxin producing selective destruction of superficial neurokinin 1 receptor bearing cells in the spinal dorsal horn. The goal of this project was to provide proof of concept data, that a single intrathecal injection of SP-SAP could safely provide effective pain relief in spontaneous bone cancer pain in companion (pet) dogs.
In a single blind, controlled study, 70 companion dogs with bone cancer pain were randomized to standard of care analgesic therapy alone (control, n=35) or intrathecal SP-SAP (20-60μg) in addition to standard of care analgesic therapy (n=35). Activity, pain scores, and videography data was collected at baseline, 2 weeks post randomization, and then monthly until death.
While the efficacy results at the 2-week post randomization point were equivocal, the outcomes evaluated beyond two weeks revealed a positive effect of SP-SAP on chronic pain management. Significantly more dogs in the control group (74%) required unblinding and adjustment in analgesic protocol or euthanasia within 6 weeks of randomization, than dogs that were treated with SP-SAP (24%; p<0.001); and overall, dogs in the control group required unblinding significantly sooner than dogs that had been treated with SP-SAP (p<0.01).
Intrathecal SP-SAP administration in dogs with bone cancer produces a time dependent anti-nociceptive effect with no evidence of development of deafferentation pain syndrome that can be seen with neurolytic therapies.
PMCID: PMC3868346  PMID: 24195949
18.  13C NMR Metabolomic Evaluation of Immediate and Delayed Mild Hypothermia in Cerebrocortical Slices After Oxygen-Glucose Deprivation 
Anesthesiology  2013;119(5):1120-1136.
Mild brain hypothermia (32°C–34°C) after human neonatal asphyxia improves neurodevelopmental outcomes. Astrocytes but not neurons have pyruvate carboxylase (PC) and an acetate uptake transporter. 13C NMR spectroscopy of rodent brain extracts after administering [1-13C]glucose and [1,2-13C]acetate can distinguish metabolic differences between glia and neurons, and tricarboxylic acid cycle (TCA cycle) entry via pyruvate dehydrogenase (PDH) and PC.
Neonatal rat cerebrocortical slices receiving a 13C-acetate/glucose mixture underwent a 45-min asphyxia simulation via oxygen-glucose-deprivation (OGD) followed by 6 h of recovery. Protocols in three groups of N = 3 experiments were identical except for temperature management. The three temperature groups were: normothermia (37°C), hypothermia (32°C for 3.75 h beginning at OGD start), and delayed hypothermia (32°C for 3.75 h, beginning 15 min after OGD start). Multivariate analysis of nuclear magnetic resonance metabolite quantifications included principal component analyses and the L1-Penalized Regularized Regression algorithm known as the Least Absolute Shrinkage and Selection Operator (LASSO).
The most significant metabolite difference (p < 0.0056) was [2-13C]glutamine’s higher final/control ratio for the Hypothermia group (1.75 ± 0.12) compared to ratios for the Delayed (1.12 ± 0.12) and Normothermia group (0.94 ± 0.06), implying a higher PC/PDH ratio for glutamine formation. LASSO found the most important metabolites associated with adenosine triphosphate preservation: [3,4-13C]glutamate—produced via PDH entry, [2-13C]taurine--an important osmolyte, and phosphocreatine. Final principal component analyses scores plots suggested separate cluster formation for the hypothermia group, but with insufficient data for statistical significance.
Starting mild hypothermia simultaneously with OGD, compared with delayed starting or no hypothermia, has higher PC throughput, suggesting that better glial integrity is one important neuroprotection mechanism of earlier hypothermia.
PMCID: PMC3890426  PMID: 23748856
19.  Exome Sequencing: One Small Step for Malignant Hyperthermia, One Giant Step for Our Specialty 
Anesthesiology  2013;119(5):1006-1008.
PMCID: PMC3980570  PMID: 24195944
20.  The effect of deep tissue incision on pH responses of afferent fibers and dorsal root ganglia innervating muscle 
Anesthesiology  2013;119(5):1186-1197.
Understanding the mechanisms underlying deep tissue pain in the postoperative period is critical to improve therapies. Using the in vitro plantar flexor digitorum brevis (FDB) muscle-nerve preparation and patch-clamp recordings from cultured dorsal root ganglia (DRG) neurons innervating incised and unincised muscle, we investigated responses to various pH changes.
Incision including the plantar FDB muscle or sham operation was made in the rat hindpaw. On postoperative day one, in vitro single fiber recording was undertaken. Based on previous studies, we recorded from at least 40 fibers per group. Also Di-I labeled DRG innervating muscle from rats undergoing incision and a sham operation were cultured and tested for acid responses using whole cell patch-clamp recordings.
The prevalence of responsive group IV afferents to lactic acid pH 6.5 in the incision group (15 of 67, 22.3%) was greater than that in the control group (2 of 35, 5.7%, p=0.022). In DRG neurons innervating muscle, incision increased mean current amplitudes of acid-evoked currents; the acid-sensing ion channel blocker, amiloride 300 μM, inhibited more than 75% of the acid-evoked current, whereas the transient receptor vanilloid receptor 1 blocker (AMG9810 1 μM) did not cause significant inhibition.
Our experiments demonstrated that incision increases the responses of FDB muscle afferent fibers to weak acid solutions, and increased acid-evoked currents in DRG innervating muscle. Our data suggest that upregulation of acid-sensing ion channels might underlie this increased chemosensitivity caused by surgery.
PMCID: PMC4028173  PMID: 23732174
21.  Using Exome Data to Identify Malignant Hyperthermia Susceptibility Mutations 
Anesthesiology  2013;119(5):1043-1053.
Malignant hyperthermia susceptibility (MHS) is a life-threatening, inherited disorder of muscle calcium metabolism, triggered by anesthetics and depolarizing muscle relaxants. An unselected cohort was screened for MHS mutations using exome sequencing. Our aim was to pilot a strategy for the RYR1 and CACNA1S genes.
Exome sequencing was performed on 870 volunteers not ascertained for MHS. Variants in RYR1 and CACNA1S were annotated using an algorithm that filtered results based on mutation type, frequency, and information in mutation databases. Variants were scored on a six-point pathogenicity scale. Medical histories and pedigrees were reviewed for malignant hyperthermia and related disorders.
We identified 70 RYR1 and 53 CACNA1S variants among 870 exomes. Sixty-three RYR1 and 41 CACNA1S variants passed the quality and frequency metrics but we excluded synonymous variants. In RYR1, we identified 65 missense mutations, one nonsense, two that affected splicing, and one non frameshift indel. In CACNA1S, 48 missense, one frameshift deletion, one splicing and one non frameshift indel were identified. RYR1 variants predicted to be pathogenic for MHS were found in three participants without medical or family histories of MHS. Numerous variants, previously described as pathogenic in mutation databases, were reclassified by us to be of unknown pathogenicity.
Exome sequencing can identify asymptomatic patients at risk for MHS, although the interpretation of exome variants can be challenging. The use of exome sequencing in unselected cohorts is an important tool to understand the prevalence and penetrance of MHS, a critical challenge for the field.
PMCID: PMC4077354  PMID: 24195946
22.  Exome Sequencing Reveals Novel Rare Variants in the Ryanodine Receptor and Calcium Channel Genes in Malignant Hyperthermia Families 
Anesthesiology  2013;119(5):1054-1065.
About half of malignant hyperthermia (MH) cases are associated with skeletal muscle ryanodine receptor 1 (RYR1) and calcium channel, voltage-dependent, L type, α1S subunit (CACNA1S) gene mutations, leaving many with an unknown cause. We chose to apply a sequencing approach to uncover causal variants in unknown cases. Sequencing the exome, the protein-coding region of the genome, has power at low sample sizes and identified the cause of over a dozen Mendelian disorders.
We considered four families with multiple MH cases but in whom no mutations in RYR1 and CACNA1S had been identified by Sanger sequencing of complementary DNA. Exome sequencing of two affecteds per family, chosen for maximum genetic distance, were compared. Variants were ranked by allele frequency, protein change, and measures of conservation among mammals to assess likelihood of causation. Finally, putative pathogenic mutations were genotyped in other family members to verify cosegregation with MH.
Exome sequencing revealed 1 rare RYR1 nonsynonymous variant in each of 3 families (Asp1056His, Val2627Met, Val4234Leu), and 1 CACNA1S variant (Thr1009Lys) in a 4th family. These were not seen in variant databases or in our control population sample of 5379 exomes. Follow-up sequencing in other family members verified cosegregation of alleles with MH.
Using both exome sequencing and allele frequency data from large sequencing efforts may aid genetic diagnosis of MH. In our sample, it was more sensitive for variant detection in known genes than Sanger sequencing of complementary DNA, and allows for the possibility of novel gene discovery.
PMCID: PMC4115638  PMID: 24013571
23.  Sleeping to survive? The impact of volatile anesthetics on mortality in sepsis 
Anesthesiology  2013;119(4):10.1097/ALN.0b013e3182a2a3a4.
PMCID: PMC3823825  PMID: 23867233
24.  Cyclosporine-inhibitable Blood-Brain Barrier Drug Transport Influences Clinical Morphine Pharmacodynamics 
Anesthesiology  2013;119(4):10.1097/ALN.0b013e3182a05bd3.
The blood-brain barrier is richly populated by active influx and efflux transporters influencing brain drug concentrations. Morphine, a drug with delayed clinical onset, is a substrate for the efflux transporter P-glycoprotein in vitro and in animals. This investigation tested whether morphine is a transporter substrate in humans.
Fourteen healthy volunteers received morphine (0.1 mg/kg, 1 h intravenous infusion) in a crossover study after nothing (control) or the validated P-glycoprotein inhibitor cyclosporine (5 mg/kg, 2 h infusion). Plasma and urine morphine and morphine glucuronide metabolite concentrations were measured by mass spectrometry. Morphine effects were measured by miosis and analgesia.
Cyclosporine minimally altered morphine disposition, increasing the area under the plasma morphine concentration versus time curve to 100 ± 21 versus 85 ± 24 ng/ml•hr (p < 0.05) without changing maximum plasma concentration. Cyclosporine enhanced (3.2 ± 0.9 vs. 2.5 ± 1.0 mm peak) and prolonged miosis, and increased the area under the miosis-time curve (18 ± 9 vs. 11 ± 5 mm-hr), plasma-effect site transfer rate constant (ke0, median 0.27 vs. 0.17 hr−1), and maximum calculated effect site morphine concentration (11.5 ± 3.7 vs. 7.6 ± 2.9 ng/ml) (all p < 0.05). Analgesia testing was confounded by cyclosporine-related pain.
Morphine is a transporter substrate at the human blood-brain barrier. Results suggest a role for P-glycoprotein or other efflux transporters in brain morphine access, although the magnitude of the effect is small, and unlikely to be a major determinant of morphine clinical effects. Efflux may explain some variability in clinical morphine effects.
PMCID: PMC3823830  PMID: 23851346
25.  Real-time Closed-loop Control in a Rodent Model of Medically-induced Coma Using Burst Suppression 
Anesthesiology  2013;119(4):10.1097/ALN.0b013e31829d4ab4.
A medically-induced coma is an anesthetic state of profound brain inactivation created to treat status epilepticus and to provide cerebral protection following traumatic brain injuries. We hypothesized that a closed-loop anesthetic delivery system could automatically and precisely control the electroencephalogram state of burst suppression and efficiently maintain a medically-induced coma.
In six rats, we implemented a closed-loop anesthetic delivery system for propofol consisting of: a computer-controlled pump infusion, a two-compartment pharmacokinetics model defining propofol’s electroencephalogram effects, the burst suppression probability algorithm to compute in real time from the electroencephalogram the brain’s burst suppression state, an on-line parameter estimation procedure and a proportional-integral controller. In the control experiment each rat was randomly assigned to one of the six burst suppression probability target trajectories constructed by permuting the burst suppression probability levels of 0.4, 0.65 and 0.9 with linear transitions between levels.
In each animal the controller maintained approximately 60 min of tight, real-time control of burst suppression by tracking each burst suppression probability target level for 15 min and two between-level transitions for 5 to 10 min. The posterior probability that the closed-loop anesthetic delivery system was reliable across all levels was 0.94 [95% confidence interval; (0.77 to 1.00) n = 18] and that the system was accurate was 1.00 [95% confidence interval; (0.84 to 1.00) n = 18].
Our findings establish the feasibility of using a closed-loop anesthetic delivery systems to achieve in real-time reliable and accurate control of burst suppression in rodents and suggest a paradigm to precisely control medically-induced coma in patients.
PMCID: PMC3857134  PMID: 23770601

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