Bispectral Index Changes Following Boluses of Commonly Used Intravenous Medications During Volatile Anesthesia Identified From Retrospective Data.

BACKGROUND: Although patients are commonly monitored for depth of anesthesia, it is unclear to what extent administration of intravenous anesthetic medications may affect calculated bispectral (BIS) index values under general anesthesia. METHODS: In a retrospective analysis of electronic anesthesia records from an academic medical center, we examined BIS index changes associated with 14 different intravenous medications, as administered in routine practice, during volatile-based anesthesia using a novel screening approach. Discrete-time windows were identified in which only a single drug bolus was administered, and subsequent changes in the BIS index, concentration of volatile anesthetic, and arterial pressure were analyzed. Our primary outcome was change in BIS index, following drug administration. Adjusted 95% confidence intervals were compared to predetermined thresholds for clinical significance. Secondary sensitivity analyses examined the same outcomes, with available data separated according to differences in baseline volatile anesthetic concentrations, doses of the administered medications, and length of time window. RESULTS: The study cohort was comprised of data from 20,170 distinct cases, 54.7% of patients were men, with a median age of 55. In the primary analysis, ketamine at a median dose of 20 mg was associated with a median (confidence limits) increase in BIS index of 3.8 (2.5-5.0). Midazolam (median dose 2 mg) was associated with a median decrease in BIS index of 3.0 (1.5-4.5). Neither of these drug administrations occurred during time periods associated with changes in volatile anesthetic concentration. Analysis for dexmedetomidine was confounded by concomitant decreases in volatile anesthetic concentration. No other medication analyzed, including propofol and common opioids, was associated with a significant change in BIS index. Secondary analyses revealed that similar BIS index changes occurred when midazolam and ketamine were administered at different volatile anesthetic concentrations and different doses, and these changes persisted 11 to 20 minutes postadministration. CONCLUSIONS: Modest, but persistent changes in BIS index occurred following doses of ketamine (increase) and midazolam (decrease) during periods of stable volatile anesthetic administration.

Preoperative Identification of Patient-Dependent Blood Pressure Targets Associated With Low Risk of Intraoperative Hypotension During Noncardiac Surgery.

BACKGROUND: Intraoperative hypotension (IOH) is strongly linked to organ system injuries and postoperative death. Blood pressure itself is a powerful predictor of IOH; however, it is unclear which pressures carry the lowest risk and may be leveraged to prevent subsequent hypotension. Our objective was to develop a model that predicts, before surgery and according to a patient's unique characteristics, which intraoperative mean arterial pressures (MAPs) between 65 and 100 mm Hg have a low risk of IOH, defined as an MAP <65 mm Hg, and may serve as testable hemodynamic targets to prevent IOH. METHODS: Adult, noncardiac surgeries under general anesthesia at 2 tertiary care hospitals of the University of Pittsburgh Medical Center were divided into training and validation cohorts, then assigned into smaller subgroups according to preoperative risk factors. Primary outcome was hypotension risk, defined for each intraoperative MAP value from 65 to 100 mm Hg as the proportion of a value's total measurements followed by at least 1 MAP <65 mm Hg within 5 or 10 minutes, and calculated for all values in each subgroup. Five models depicting MAP-associated IOH risk were compared according to best fit across subgroups with proportions whose confidence interval was <0.05. For the best fitting model, (1) performance was validated, (2) low-risk MAP targets were identified according to applied benchmarks, and (3) preoperative risk factors were evaluated as predictors of model parameters. RESULTS: A total of 166,091 surgeries were included, with 121,032 and 45,059 surgeries containing 5.4 million and 1.9 million MAP measurements included in the training and validation sets, respectively. Thirty-six subgroups with at least 21 eligible proportions (confidence interval <0.05) were identified, representing 92% and 94% of available MAP measurements, respectively. The exponential with theta constant model demonstrated the best fit (weighted sum of squared error 0.0005), and the mean squared error of hypotension risk per MAP did not exceed 0.01% in validation testing. MAP targets ranged between 69 and 90 mm Hg depending on the subgroup and benchmark used. Increased age, higher American Society of Anesthesiologists physical status, and female sexindependently predicted ( P < .05) hypotension risk curves with less rapid decay and higher plateaus. CONCLUSIONS: We demonstrate that IOH risk specific to a given MAP is patient-dependent, but predictable before surgery. Our model can identify intraoperative MAP targets before surgery predicted to reduce a patient's exposure to IOH, potentially allowing clinicians to develop more personalized approaches for managing hemodynamics.

Impact of Neuraxial Versus General Anesthesia on Discharge Destination in Patients Undergoing Primary Total Hip and Total Knee Replacement.

BACKGROUND: Total knee replacement (TKR) and total hip replacement (THR) are 2 of the most common orthopedic surgical procedures in the United States. These procedures, with fairly low mortality rates, incur significant health care costs, with almost 40% of the costs associated with post acute care. We assessed the impact of general versus neuraxial anesthesia on discharge destination and 30-day readmissions in patients who underwent total knee and hip replacement in our health system. METHODS: This was a retrospective cohort study of 24,684 patients undergoing total knee or hip replacement in 13 hospitals of a large health care network. Following propensity score matching, we studied the impact of type of anesthetic technique on discharge destination (primary outcome) and postoperative complications including readmissions in 8613 patients who underwent THR and 13,004 patients for TKR. RESULTS: Our results showed that in patients undergoing THR and TKR, neuraxial anesthesia is associated with higher odds of being discharged from hospital to home versus other facilities compared to general anesthesia (odds ratio [OR] = 1.63, 95% confidence interval [CI], 1.52-1.76; P < .01) and (OR = 1.58, 95% CI, 1.49-1.67; P < .01), respectively. CONCLUSIONS: Our results suggest an association between use of neuraxial anesthesia for total joint arthroplasty and a higher probability of discharge to home and a reduction in readmissions.

Retrospective analysis of cases of intraoperative awareness in a large multi-hospital health system reported in the early postoperative period.

BACKGROUND: Awareness with recall under general anesthesia remains a rare but important issue that warrants further study. METHODS: We present a series of seven cases of awareness that were identified from provider-reported adverse event data from the electronic anesthesia records of 647,000 general anesthetics. RESULTS: The low number of identified cases suggests an under-reporting bias. Themes that emerge from this small series can serve as important reminders to anesthesia providers to ensure delivery of an adequate anesthetic for each patient. Commonalities between a majority of our identified anesthetic awareness cases include: obesity, use of total intravenous anesthesia, use of neuromuscular blockade, and either a lack of processed electroencephalogram (EEG) monitoring or documented high depth of consciousness index values. An interesting phenomenon was observed in one case, where adequately-dosed anesthesia was delivered without technical issue, processed EEG monitoring was employed, and the index value suggested an adequate depth of consciousness throughout the case. CONCLUSIONS: Provider-reported adverse event data in the immediate post-operative period are likely insensitive for detecting cases of intraoperative awareness. Though causation cannot firmly be established from our data, themes identified in this series of cases of awareness with recall under general anesthesia provide important reminders for anesthesia providers to maintain vigilance in monitoring depth and dose of anesthesia, particularly with total intravenous anesthesia.

The triple variable index combines information generated over time from common monitoring variables to identify patients expressing distinct patterns of intraoperative physiology.

BACKGROUND: Mean arterial pressure (MAP), bispectral index (BIS), and minimum alveolar concentration (MAC) represent valuable, yet dynamic intraoperative monitoring variables. They provide information related to poor outcomes when considered together, however their collective behavior across time has not been characterized. METHODS: We have developed the Triple Variable Index (TVI), a composite variable representing the sum of z-scores from MAP, BIS, and MAC values that occur together during surgery. We generated a TVI expression profile, defined as the sequential TVI values expressed across time, for each surgery where concurrent MAP, BIS, and MAC monitoring occurred in an adult patient (≥18 years) at the University of Pittsburgh Medical Center between January and July 2014 (n = 5296). Patterns of TVI expression were identified using k-means clustering and compared across numerous patient, procedure, and outcome characteristics. TVI and the triple low state were compared as prediction models for 30-day postoperative mortality. RESULTS: The median frequency MAP, BIS, and MAC were recorded was one measurement every 3, 5, and 5 min. Three expression patterns were identified: elevated, mixed, and depressed. The elevated pattern displayed the highest average MAP, BIS, and MAC values (86.5 mmHg, 45.3, and 0.98, respectively), while the depressed pattern displayed the lowest values (76.6 mmHg, 38.0, 0.66). Patterns (elevated, mixed, depressed) were distinct across the following characteristics: average patient age (52, 53, 54 years), American Society of Anesthesiologists Physical Status 4 (6.7, 16.1, 27.3%) and 5 (0.1, 0.6, 1.6%) categories, cardiac (2.2, 6.5, 16.1%) and emergent (5.8, 10.5, 12.8%) surgery, cardiopulmonary bypass use (0.3, 2.6, 9.8%), intraoperative medication administration including etomidate (3.0, 7.3, 12.6%), hydromorphone (47.6, 26.3, 25.2%), ketamine (11.2, 4.6, 3.0%), dexmedetomidine (18.4, 16.6, 13.6%), phenylephrine (74.0, 74.8, 83.0), epinephrine (2.0, 6.0, 18.0%), norepinephrine (2.4, 7.5, 21.2%), vasopressin (3.4, 7.6, 21.0%), succinylcholine (74.0, 69.0, 61.9%), intraoperative hypotension (28.8, 33.0, 52.3%) and the triple low state (9.4, 30.3, 80.0%) exposure, and 30-day postoperative mortality (0.8, 2.7, 5.6%). TVI was a better predictor of patients that died or survived in the 30 days following surgery compared to cumulative triple low state exposure (AUC 0.68 versus 0.62, p < 0.05). CONCLUSIONS: Surgeries that share similar patterns of TVI expression display distinct patient, procedure, and outcome characteristics.

Phospholipase C-gamma1 is involved in signaling the activation by high NaCl of the osmoprotective transcription factor TonEBP/OREBP.

High NaCl elevates activity of the osmoprotective transcription factor TonEBP/OREBP by increasing its phosphorylation, transactivating activity, and localization to the nucleus. We investigated the possible role in this activation of phospholipase C-gamma1 (PLC-gamma1), which has a predicted binding site at TonEBP/OREBP-phospho-Y143. We find the following. (i) Activation of TonEBP/OREBP transcriptional activity by high NaCl is reduced in PLC-gamma1 null cells and in HEK293 cells in which PLC-gamma1 is knocked down by a specific siRNA. (ii) High NaCl increases phosphorylation of TonEBP/OREBP at Y143. (iii) Wild-type PLC-gamma1 coimmunoprecipitates with wild-type TonEBP/OREBP but not TonEBP/OREBP-Y143A, and the coimmunoprecipitation is increased by high NaCl. (iv) PLC-gamma1 is part of the protein complex that associates with TonEBP/OREBP at its DNA binding site. (v) Knockdown of PLC-gamma1 or overexpression of a PLC-gamma1-SH3 deletion mutant reduces high NaCl-dependent TonEBP/OREBP transactivating activity. (vi) Nuclear localization of PLC-gamma1 is increased by high NaCl. (vii) High NaCl-induced nuclear localization of TonEBP/OREBP is reduced if cells lack PLC-gamma1, if PLC-gamma1 mutated in its SH2C domain is overexpressed, or if Y143 in TonEBP/OREBP is mutated to alanine. (viii) Expression of recombinant PLC-gamma1 restores nuclear localization of wild-type TonEBP/OREBP in PLC-gamma1 null cells but not of TonEBP/OREBP-Y143A. (ix) The PLC-gamma1 phospholipase inhibitor U72133 inhibits nuclear localization of TonEBP/OREBP but not the increase of its transactivating activity. We conclude that, when NaCl is elevated, TonEBP/OREBP becomes phosphorylated at Y143, resulting in binding of PLC-gamma1 to that site, which contributes to TonEBP/OREBP transcriptional activity, transactivating activity, and nuclear localization.

CHD7 targets active gene enhancer elements to modulate ES cell-specific gene expression.

CHD7 is one of nine members of the chromodomain helicase DNA-binding domain family of ATP-dependent chromatin remodeling enzymes found in mammalian cells. De novo mutation of CHD7 is a major cause of CHARGE syndrome, a genetic condition characterized by multiple congenital anomalies. To gain insights to the function of CHD7, we used the technique of chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-Seq) to map CHD7 sites in mouse ES cells. We identified 10,483 sites on chromatin bound by CHD7 at high confidence. Most of the CHD7 sites show features of gene enhancer elements. Specifically, CHD7 sites are predominantly located distal to transcription start sites, contain high levels of H3K4 mono-methylation, found within open chromatin that is hypersensitive to DNase I digestion, and correlate with ES cell-specific gene expression. Moreover, CHD7 co-localizes with P300, a known enhancer-binding protein and strong predictor of enhancer activity. Correlations with 18 other factors mapped by ChIP-seq in mouse ES cells indicate that CHD7 also co-localizes with ES cell master regulators OCT4, SOX2, and NANOG. Correlations between CHD7 sites and global gene expression profiles obtained from Chd7(+/+), Chd7(+/-), and Chd7(-/-) ES cells indicate that CHD7 functions at enhancers as a transcriptional rheostat to modulate, or fine-tune the expression levels of ES-specific genes. CHD7 can modulate genes in either the positive or negative direction, although negative regulation appears to be the more direct effect of CHD7 binding. These data indicate that enhancer-binding proteins can limit gene expression and are not necessarily co-activators. Although ES cells are not likely to be affected in CHARGE syndrome, we propose that enhancer-mediated gene dysregulation contributes to disease pathogenesis and that the critical CHD7 target genes may be subject to positive or negative regulation.

CHD7 functions in the nucleolus as a positive regulator of ribosomal RNA biogenesis.

De novo mutation of the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7) is the primary cause of CHARGE syndrome, a complex developmental disorder characterized by the co-occurrence of a specific set of birth defects. Recent studies indicate that CHD7 functions as a transcriptional regulator in the nucleoplasm. Here, we report based on immunofluorescence and western blotting of subcellular fractions that CHD7 is also constitutively localized to the nucleolus, the site of rRNA transcription. Standard chromatin immunoprecipitation (ChIP) assays indicate that CHD7 physically associates with rDNA, a result that is also observable upon alignment of whole-genome CHD7 ChIP coupled with massively parallel DNA sequencing data to the rDNA reference sequence. ChIP-chop analyses demonstrate that CHD7 specifically associates with hypomethylated, active rDNA, suggesting a role as a positive regulator of rRNA synthesis. Consistent with this hypothesis, siRNA-mediated depletion of CHD7 results in hypermethylation of the rDNA promoter and a concomitant reduction of 45S pre-rRNA levels. Accordingly, cells overexpressing CHD7 show increased levels of 45S pre-rRNA compared with control cells. Depletion of CHD7 also reduced cell proliferation and protein synthesis. Lastly, compared with wild-type ES cells, the levels of 45S pre-rRNA are reduced in both Chd7(+/-) and Chd7(-/-) mouse ES cells, as well as in Chd7(-/-) whole mouse embryos and multiple tissues dissected from Chd7(+/-) embryos. Together with previously published studies, these results indicate that CHD7 dually functions as a regulator of both nucleoplasmic and nucleolar genes and provide a novel avenue for investigation into the pathogenesis of CHARGE syndrome.

Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies.

We developed a strategy to introduce epitope tag-encoding DNA into endogenous loci by homologous recombination-mediated 'knock-in'. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation-microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.

Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns.

CHD7 is a member of the chromodomain helicase DNA binding domain family of ATP-dependent chromatin remodeling enzymes. De novo mutation of the CHD7 gene is a major cause of CHARGE syndrome, a genetic disease characterized by a complex constellation of birth defects (Coloboma of the eye, Heart defects, Atresia of the choanae, severe Retardation of growth and development, Genital abnormalities, and Ear abnormalities). To gain insight into the function of CHD7, we mapped the distribution of the CHD7 protein on chromatin using the approach of chromatin immunoprecipitation on tiled microarrays (ChIP-chip). These studies were performed in human colorectal carcinoma cells, human neuroblastoma cells, and mouse embryonic stem (ES) cells before and after differentiation into neural precursor cells. The results indicate that CHD7 localizes to discrete locations along chromatin that are specific to each cell type, and that the cell-specific binding of CHD7 correlates with a subset of histone H3 methylated at lysine 4 (H3K4me). The CHD7 sites change concomitantly with H3K4me patterns during ES cell differentiation, suggesting that H3K4me is part of the epigenetic signature that defines lineage-specific association of CHD7 with specific sites on chromatin. Furthermore, the CHD7 sites are predominantly located distal to transcription start sites, most often contained within DNase hypersensitive sites, frequently conserved, and near genes expressed at relatively high levels. These features are similar to those of gene enhancer elements, raising the possibility that CHD7 functions in enhancer mediated transcription, and that the congenital anomalies in CHARGE syndrome are due to alterations in transcription of tissue-specific genes normally regulated by CHD7 during development.

Proteomic identification of proteins associated with the osmoregulatory transcription factor TonEBP/OREBP: functional effects of Hsp90 and PARP-1.

Hypertonicity (e.g., high NaCl) activates the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP), increasing transcription of protective genes. In the present studies, by stably expressing amino acids 1-547 of TonEBP/OREBP in HEK 293 cells and immunoprecipitating it plus associated proteins from the nuclei of cells exposed to high NaCl, we identify 14 proteins that are physically associated with TonEBP/OREBP. The associated proteins fall into several classes: 1) DNA-dependent protein kinase, both its catalytic subunit and regulatory subunit, Ku86; 2) RNA helicases, namely RNA helicase A, nucleolar RNA helicase II/Gu, and DEAD-box RNA helicase p72; 3) small or heterogeneous nuclear ribonucleoproteins (snRNPs or hnRNPs), namely U5 snRNP-specific 116 kDa protein, U5 snRNP-specific 200 kDa protein, hnRNP U, hnRNP M, hnRNP K, and hnRNP F; 4) heat shock proteins, namely Hsp90beta and Hsc70; and 5) poly(ADP-ribose) polymerase-1 (PARP-1). We confirm identification of most of the proteins by Western analysis and also demonstrate by electrophoretic mobility-shift assay that they are present in the large complex that binds specifically along with TonEBP/OREBP to its cognate DNA element. In addition, we find that PARP-1 and Hsp90 modulate TonEBP/OREBP activity. PARP-1 expression reduces TonEBP/OREBP transcriptional activity and the activity of its transactivating domain. Hsp90 enhances those activities and sustains the increased abundance of TonEBP/OREBP protein in cells exposed to high NaCl.