Emergence Electroencephalography in an Unresponsiveness Geriatric Patient in the Postanesthesia Care Unit: A Case Report.

Incomplete neurological awakening manifested as aberrant patterns of electroencephalography (EEG) at emergence may be responsible for an unresponsive patient in the postanesthesia care unit (PACU). We describe a case of an individual who remained unresponsive but awake in the PACU. Retrospective, intraoperative EEG analysis showed low alpha power and a sudden shift from deep delta to arousal preextubation. We explored parallels with diminished motivation disorders and anesthesia-induced sleep paralysis due to imbalances in anesthetic drug sensitivity between brain regions. Our findings highlight the relevance of end-anesthesia EEG patterns in diagnosing delayed awakening.

Preliminary pharmacokinetics and patient experience of jet-injected dexmedetomidine in healthy adults.

Jet injection is a drug delivery system without a needle. A compressed liquid drug formulation pierces the skin, depositing the drug into the subcutaneous or intramuscular tissues. We investigated the pharmacokinetics and patient experience of dexmedetomidine administered using jet injection in six healthy adult study participants. This needleless jet injection device was used to administer dexmedetomidine 0.5 µg/kg to the subcutaneous tissues overlying the deltoid muscle. Serum concentrations of dexmedetomidine were assayed at approximately 5 minutes, 15 minutes, 30 minutes, 1 hour and 4 hours after administration. Pharmacokinetic interrogation of concentration time profiles estimated an absorption half time for jet-injected dexmedetomidine of 21 minutes (coefficient of variation 69.4%) with a relative bioavailability assumed unity. In our samples the measured median peak (range) concentration was 0.164 µg/l (0.011-0.325 µg/l), observed in the sample taken at a median (range) of 13.5 minutes (11-30 minutes). The Richmond agitation sedation scale was used to assess the sedative effect, and scored 0 (alert and calm) or -1 (drowsy) in all participants. Five of the six participants stated they would prefer jet injection to needle injection in the future and one had no preference. The findings suggest that the use of a larger dose (>2 µg/kg) would be required to achieve the clinically relevant target concentration of 1 µg/l necessary to achieve deeper sedation (Richmond agitation sedation scale ≤3).

Seeing is Believing: Chasing Sevoflurane Vapor Trails.

PURPOSE: Sevoflurane is an inhalational general anesthetic that has been used recently to treat chronic, painful lesions, reportedly supporting analgesia and wound healing. The potential for repeated exposure to off-gassed sevoflurane vapor, especially outside the air-conditioned operating theatre environment, is of some concern. DESIGN: This paper explores the qualitative and quantitative pathing of off-gassed sevoflurane from a topically applied liquid source. METHODS: Using a small, unventilated test-box (total volume 0.5 m3) with infra-red imaging and gas-analysing, we investigated the spatial distribution of sevoflurane vapor following complete vaporization of a 20 mL liquid sample. Utilizing the infra-red absorption of sevoflurane, it was possible to visualize (as an apparent reduction in temperature) the streaming path of the sevoflurane vapor. Sevoflurane levels (%) in the test-box were measured using an infra-red gas analyzer. FINDINGS: In keeping with its higher density than air, sevoflurane vapor was seen to "waterfall" from the liquid source and accumulate in the bottom of the test-box. Sevoflurane vapor concentration was minimal above the liquid source. When extrapolated to a larger (unventilated) room, we estimate that the sevoflurane concentration would be less than 10 ppm one centimetre above the liquid pool. With vacuum extraction, these levels would be even lower. CONCLUSIONS: Due to sevoflurane's tendency to accumulate on the floor, it is concluded that topical application of liquid sevoflurane posses virtually no risk to off-gas exposure in unventilated spaces.

Tissue oxygen partial pressure as a viability metric for ex vivo brain tissue slices.

BACKGROUND: Despite the prevalent use of the ex vivo brain slice preparation in neurophysiology research, a reliable method for judging tissue viability - and thus suitability of a slice for inclusion in an experiment - is lacking. The utility of indirect electrophysiological measures of tissue health is model-specific and needs to be used cautiously. In this study, we verify a more direct test of slice viability, based on tissue oxygen consumption rate. NEW METHOD: We hypothesised that the minimum intra-slice partial pressure of oxygen (pO2min) would correlate with tissue oxygen consumption rate, providing an accessible method for reliably assessing tissue viability status. Using mouse brain cortex slices, we measured tissue oxygen consumption rate using a Fick's law diffusion-consumption model applied to full intra-tissue pO2 profiles and compared this to pO2min and 2,3,5-triphenol tetrazolium chloride (TTC) viability staining. RESULTS: Tissue pO2min correlated strongly with oxygen consumption rate in both neurophysiological active and quiescent tissue (in "no-magnesium" and "normal" artificial cerebrospinal fluid, respectively) (R2 =49.7% and 42.1%, respectively). Both correlated with TTC viability stain. Oxygen consumption rate was positively related to the frequency of seizure-like event activity in no-magnesium artificial cerebrospinal fluid (R2 = 44.8%). COMPARISON WITH EXISTING METHODS: While measurement of tissue oxygen levels and oxygen consumption is not new, intra-tissue pO2min is a novel approach to assess brain slice viability. CONCLUSION: The results confirm that tissue oxygen minimum pO2min is a robust metric for estimating tissue viability status - the lower the pO2min, the healthier the tissue.

Determination of Krogh Coefficient for Oxygen Consumption Measurement from Thin Slices of Rodent Cortical Tissue Using a Fick's Law Model of Diffusion.

To investigate the impact of experimental interventions on living biological tissue, ex vivo rodent brain slices are often used as a more controllable alternative to a live animal model. However, for meaningful results, the biological sample must be known to be healthy and viable. One of the gold-standard approaches to identifying tissue viability status is to measure the rate of tissue oxygen consumption under specific controlled conditions. Here, we work with thin (400 µm) slices of mouse cortical brain tissue which are sustained by a steady flow of oxygenated artificial cerebralspinal fluid (aCSF) at room temperature. To quantify tissue oxygen consumption (Q), we measure oxygen partial pressure (pO2) as a function of probe depth. The curvature of the obtained parabolic (or parabola-like) pO2 profiles can be used to extract Q, providing one knows the Krogh coefficient Kt, for the tissue. The oxygen trends are well described by a Fick's law diffusion-consumption model developed by Ivanova and Simeonov, and expressed in terms of ratio (Q/K), being the rate of oxygen consumption in tissue divided by the Krogh coefficient (oxygen diffusivity × oxygen solubility) for tissue. If the fluid immediately adjacent to the tissue can be assumed to be stationary (i.e., nonflowing), one may invoke conservation of oxygen flux K·(∂P/∂x) across the interface to deduce (Kt/Kf), the ratio of Krogh coefficients for tissue and fluid. Using published interpolation formulas for the effect of salt content and temperature on oxygen diffusivity and solubility for pure water, we estimate Kf, the Krogh coefficient for aCSF, and hence deduce the Kt coefficient for tissue. We distinguish experimental uncertainty from natural biological variability by using pairs of repeated profiles at the same tissue location. We report a dimensionless Krogh ratio (Kt/Kf)=0.562±0.088 (mean ± SD), corresponding to a Krogh coefficient Kt=(1.29±0.21)×10-14 mol/(m·s·Pa) for mouse cortical tissue at room temperature, but acknowledge the experimental limitation of being unable to verify that the fluid boundary layer is truly stationary. We compare our results with those reported in the literature, and comment on the challenges and ambiguities caused by the extensive use of 'biologically convenient' non-SI units for tissue Krogh coefficient.

Deconstructing delirium in the post anaesthesia care unit.

The course of neuro-cognitive recovery following anaesthesia and surgery is distinctive and poorly understood. Our objective was to identify patterns of neuro-cognitive recovery of the domains routinely assessed for delirium diagnosis in the post anaesthesia care unit (PACU) and to compare them to the cognitive recovery patterns observed in other studies; thereby aiding in the identification of pathological (high risk) patterns of recovery in the PACU. We also compared which of the currently available tests (3D-CAM, CAM-ICU, and NuDESC) is the best to use in PACU. This was a post hoc secondary analysis of data from the Alpha Max study which involved 200 patients aged over 60 years, scheduled for elective surgery under general anaesthesia lasting more than 2 h. These patients were assessed for delirium at 30 min following arrival in the PACU, if they were adequately arousable (Richmond Agitation Sedation Score ≥ -2). All tests for delirium diagnosis (3D-CAM, CAM-ICU, and NuDESC) and the sub-domains assessed were compared to understand temporal recovery of neurocognitive domains. These data were also analysed to determine the best predictor of PACU delirium. We found the incidence of PACU delirium was 35% (3D-CAM). Individual cognitive domains were affected differently. Few individuals had vigilance deficits (6.5%, n = 10 CAM-ICU) or disorganized thinking (19% CAM-ICU, 27.5% 3D-CAM), in contrast attention deficits were common (72%, n = 144) and most of these patients (89.5%, n = 129) were not sedated (RASS ≥ -2). CAM-ICU (27%) and NuDESC (52.8%) detected fewer cases of PACU delirium compared to 3D-CAM. In conclusion, return of neurocognitive function is a stepwise process; Vigilance and Disorganized Thinking are the earliest cognitive functions to return to baseline and lingering deficits in these domains could indicate an abnormal cognitive recovery. Attention deficits are relatively common at 30 min in the PACU even in individuals who appear to be awake. The 3D CAM is a robust test to check for delirium in the PACU.

The general anaesthetic propofol prevents cerebrocortical potentiation in neocortical mouse brain slices.

Propofol is well known to cause amnesia independent of its sedative effect. Memory consolidation processes in the hippocampus have been proposed as a target - however the neural substrates for propofol's amnesic actions remain understudied and poorly described. In particular, the potential role of the cerebral cortex has not been investigated. As an in vitro experimental model of cortical memory consolidation, potentiated cerebral cortex evoked responses were generated in mouse neocortical slices using high frequency (20 Hz) stimulation to layer IV cortical grey matter or subcortical white matter. In separate experiments, slices were pretreated with propofol at two concentrations, 2 µg/mL and 4 µg/mL, to determine the effect of clinically relevant propofol levels on the potentiation response. Only grey matter stimulation induced a significant and lasting increase in cortical evoked potential amplitude in the drug-free condition. Propofol at 2 µg/mL completely inhibited cortical evoked response potentiation, while the 4 µg/mL concentration caused a small but significant depressant effect consequent to the high frequency stimulation. These findings support the hypothesis that propofol disrupts memory consolidation and actively facilitates memory decay in the cerebral cortex. The results further highlight the importance of the cerebral cortex in the early phase of long term memory consolidation.

Non-NMDA Mechanisms of Analgesia in Ketamine Analogs.

Despite 50 years of clinical use and experimental endeavor the anesthetic, analgesic, and psychomimetic effects of ketamine remain to be fully elucidated. While NMDA receptor antagonism has been long held as ketamine's fundamental molecular action, interrogation of bespoke ketamine analogs with known absent NMDA binding, yet profound anesthetic and analgesia fingerprints, suggests alternative targets are responsible for these effects. Herein we describe experimental findings utilizing such analogs as probes to explore ketamine-based analgesic molecular targets. We have focused on two-pore potassium leak channels, identifying TWIK channels as a rational target to pursue further. While the totality of ketamine's mechanistic action is yet to be fully determined, these investigations raise the intriguing prospect of separating out analgesia and anesthetic effects from ketamine's undesirable psychomimesis-and development of more specific analgesic medications.

A systematic exploration of local network state space in neocortical mouse brain slices.

The ex vivo cortical slice is an extremely versatile preparation, but its utility ultimately depends on understanding its limitations and functional constraints. A question for experimentalists new to the field of cortical slice electrophysiology might be - what are the different network dynamical states available to a cortical slice as a function of excitatory drive? The purpose of this study is to provide a coherent answer to this question, within the context of extracellularly recorded population field potentials. Cortical slices (400 µm) were prepared from adult male or female C57 mice. Evoked responses were recorded within cortical layer III/IV using extracellularly positioned metal electrodes. In the first part of the study, slice excitatory drive was increased by reducing the concentration of magnesium ions in the artificial cerebrospinal fluid - and the evoked responses categorized during the transition. In the second part, each of the identified functional states were explored in greater detail with tissue perfusion conditions and excitatory drive optimised for the requisite response state. As expected, rodent cortical slices did not generate spontaneous, persistent EEG-like field potential activity. However, distinct response states (spontaneous and evoked) characterized by intermittent population bursts could be differentiated as a function of excitatory drive. Each state reflected different modes of neocortical activation: "monosynaptic" responses were brief, non-propagating activations, reflecting an inhibited cortex with sensory processing blocked; polysynaptic and epileptiform activity propagated intra-cortically, the latter reflecting a hyperactivated, hypersynchronous "seizing" cortex. Polysynaptic activity most closely resembled sensory "up states" associated with intracortical sensory processing. Understanding the functional distinction between the different cortical slice response states is the starting point for designing experiments that maximise the utility of this ex vivo model. The results and descriptions in this study should help slice experimentalists less experienced in the nuances of cortical slice neurophysiology to make informed choices about how to tailor the parameters of the model to suit the specific aims of their research.

Microalgae-based photosynthetic strategy for oxygenating avascularised mouse brain tissue - An in vitro proof of concept study.

Hypoxic brain injury is a leading cause of loss of quality of life globally for which there are currently no effective treatments. There has been increasing interest in incorporating photosynthesising agents into hypoxic tissue as a mechanism for in situ oxygen delivery, independent of vascular perfusion. To date this has not been tested in the brain. The oxygen production capacity of Chlamydomonas reinhardtii microalgal cultures was measured in artificial cerebrospinal fluid (aCSF) in benchtop assays and in cortical slices in situ. Cortical slice function was quantified by measuring the length, frequency and amplitude of seizure-like event (SLE) activity - in conventionally oxygenated aCSF, C. reinhardtii cultures, unoxygenated and deoxygenated aCSF. The possibility of direct toxic algal effects was investigated by exposing slices to cultures for 5 h. An oxygen level of 25 mg.L-1 was achieved with C. reinhardtii in no-Mg aCSF. Slice SLE function was preserved in C. reinhardtii, without the need for supplemental oxygen. In contrast, functional parameters deteriorated in unoxygenated and deoxygenated aCSF. In the former, there was a 66% reduction in SLE frequency and a 37% reduction in event amplitude. In the latter, SLE activity ceased completely. No toxic algae effects were seen in slices exposed to cultures for 5 h. These results confirm that C. reinhardtii oxygenation of aCSF can sustain cortical network activity - without tissue toxicity for the normal lifespan of an acute cortical slice. This study shows promise for the concept of photosynthesis as a mechanism for providing oxygen to rescue ischaemic avascularised brain tissue.

Electrophysiological field potential identification of an intact GABAergic system in mouse cortical slices.

In brain slice experiments there's currently no validated electrophysiological method for differentiating viability between GABAergic and glutamatergic cell populations. Here we investigated the neurophysiology of high frequency field potential activity - and its utility for probing the functional state of the GABAergic system in brain slices. Field potentials were recorded from mouse cortical slices exposed to 50 mM potassium ("elevated-K") and the induced high frequency (>20 Hz) response characterized pharmacologically. The elevated-K responses were also related to the high frequency activity imbedded in no-magnesium seizure-like events (SLE) from the same slices. The elevated-K response, comprising a transient burst of high frequency activity, was strongly GABAA-dependent. The size of the high frequency response was reduced by 71% (p = 0.001) by picrotoxin, but not significantly attenuated by either APV or CNQX. High frequency activity embedded in no-magnesium SLEs correlated with the elevated-K response. The success rate for generating an elevated-K response - and high frequency SLE activity - declined rapidly with increasing time since slicing. These findings support the hypothesis that in cortical slices, a functioning synaptic GABAergic system is evidenced by a strong high frequency component to no-magnesium SLE activity - and that the integrity of the GABAergic system degrades quicker than the excitatory glutamatergic system in this preparation.

Application of a benchtop colorimetric method for quantification of blood propofol levels.

Quantification of plasma propofol (2,6-diisopropylphenol) in the context of clinical anaesthesia is challenging because of the need for offline blood sample processing using specialised laboratory equipment and techniques. In this study we sought to refine a simple procedure using solid phase extraction and colorimetric analysis into a benchtop protocol for accurate blood propofol measurement. The colorimetric method based on the reaction of phenols (e.g. propofol) with Gibbs reagent was first tested in 10% methanol samples (n = 50) containing 0.5-6.0 µg/mL propofol. Subsequently, whole blood samples (n = 15) were spiked to known propofol concentrations and processed using reverse phase solid phase extraction (SPE) and colorimetric analysis. The standard deviation of the difference between known and measured propofol concentrations in the methanol samples was 0.11 µg/mL, with limits of agreement of - 0.21 to 0.22 µg/mL. For the blood-processed samples, the standard deviation of the difference between known and measured propofol concentrations was 0.09 µg/mL, with limits of agreement - 0.18 to 0.17 µg/mL. Quantification of plasma propofol with an error of less than 0.2 µg/mL is achievable with a simple and inexpensive benchtop method.

Cerebrospinal fluid oxygen optimisation for rescue of metabolically challenged in vitro cortical brain tissue.

Hypoxic-ischaemic brain injury is a major cause of morbidity and mortality internationally. Using an in vitro isolated cortex model, this study investigated the optimal cerebrospinal fluid oxygenation parameters for rescuing metabolically challenged cortical tissue. In particular, we asked whether maximizing oxygen content with oxygen nanobubbles could support improved tissue recovery. Mouse cortical slices were metabolically starved, followed by recovery in artificial cerebrospinal fluid (aCSF) containing different levels of dissolved oxygen ranging from mean(SD) 2(0.5) to 39(1.0) mg/L; with and without oxygen nanobubbles. Tissue recovery was assessed by quantifying and comparing the amplitude, length, high frequency content and event frequency of seizure-like events generated in no-magnesium aCSF at the beginning and end of the protocol. In general, there was improved recovery with increasing oxygen content up to 25-34 mg/L. The outcome of slices recovered in nanobubbled aCSF was no different to conventionally oxygenated slices with similar dissolved oxygen content. Dissolved oxygen content above 34 mg/L afforded no additional benefit. In conclusion, aCSF dissolved oxygen content of approximately 30 mg/L is optimal for cortical tissue recovery from metabolic starvation, which is easily achievable using conventional oxygenation methods. Oxygen in the form of nanobubbles does not appear to be readily available for tissue oxidative processes in this model.

Impact of variation in tissue preparation methodology on the functional outcome of neocortical mouse brain slices.

The in vitro cortical slice preparation is a widely used tool for electrophysiological investigation of brain neurophysiology. However, slice quality can be highly variable despite attempts to standardise practice. The purpose of this study was to determine the extent to which this variability is due to sensitivity to aspects of the preparation methodology. The study ultilised the no-magnesium seizure-like event (SLE) model and was in two parts. In the first, slice outcome was quantified following a standardised hypoxic-ischaemic insult applied to pre-prepared slices. The hypoxia-induced changes in SLE activity were used to characterise the effect of tissue damage on commonly measured electrophysiological variables. In the second, multivariate data associated with the slice preparation methodology was collected and related to tissue outcome, according to the same SLE characteristics. Hypoxia-ischaemia damage was reflected most strongly in a reduction in SLE amplitude, inter-event frequency and intra-event high frequency activity. The number of active locations was also markedly reduced. In the second part of the study, between 21% and 47% of outcome variation was attributable to variability in the methodological parameters tested. Most important were slice position from the tissue block, depth of slice submersion during recording and the experience level of the experimenter. The latter showed a paradoxical relationship between inexperience, heightened SLE amplitude and reduced spatial viability. While much of the variation in slice outcome remained unexplained, the factors shown to correlate with tissue viability will aid slice experimentalists in preparing tissue of optimal quality for electrophysiological investigation. In particular, using population event amplitude as the sole criterion of slice quality in the no-magnesium model is overly simplistic and must be viewed in the context of spatial activity.

Relationship between artificial cerebrospinal fluid oxygenation, slice depth and tissue performance in submerged brain slice experiments.

One of the challenges for slice experimentalists is achieving optimal tissue oxygenation. One area that has not been addressed in submerged slices is the relationship between oxygenation of the artificial cerebrospinal fluid, slice depth and tissue performance. In this study we varied the depth of slice submersion, measured the oxygen profile in the solution and related these to slice activity in the form of spontaneous population events. While the oxygen profile curves were qualitatively similar (peaking approximately 1.7 mm below the solution surface), the average oxygen content was highly variable and correlated strongly with slice depth (R2 = 39%, p < 0.0001). The solution oxygen content was positively correlated with the frequency of population events (R2 = 21%, p < 0.0001). Optimum oxygenation and tissue performance were achieved with slices submerged at least 1.7 mm below the solution surface. In conclusion, in brain slice experiments it is important to measure and control the depth of slice submersion to maximise tissue oxygenation and reduce output variation between trials.

A Metabolic Mechanism for Anaesthetic Suppression of Cortical Synaptic Function in Mouse Brain Slices-A Pilot Investigation.

Regulation of synaptically located ionotropic receptors is thought to be the main mechanism by which anaesthetics cause unconsciousness. An alternative explanation, which has received much less attention, is that of primary anaesthetic disruption of brain metabolism via suppression of mitochondrial proteins. In this pilot study in mouse cortical slices, we investigated the effect of disrupting cellular metabolism on tissue oxygen handling and cortical population seizure-like event (SLE) activity, using the mitochondrial complex I inhibitor rotenone, and compared this to the effects of the general anaesthetics sevoflurane, propofol and ketamine. Rotenone caused an increase in tissue oxygen (98 mmHg to 157 mmHg (p < 0.01)) before any measurable change in SLE activity. Thereafter, tissue oxygen continued to increase and was accompanied by a significant and prolonged reduction in SLE root mean square (RMS) activity (baseline RMS of 1.7 to 0.7 µV, p < 0.001) and SLE frequency (baseline 4.2 to 0.4 events/min, p = 0.001). This temporal sequence of effects was replicated by all three anaesthetic drugs. In conclusion, anaesthetics with differing synaptic receptor mechanisms all effect changes in tissue oxygen handling and cortical network activity, consistent with a common inhibitory effect on mitochondrial function. The temporal sequence suggests that the observed synaptic depression-as seen in anaesthesia-may be secondary to a reduction in cellular metabolic capacity.

Structure-Activity Relationships for the Anaesthetic and Analgaesic Properties of Aromatic Ring-Substituted Ketamine Esters.

A series of benzene ring substituted ketamine N-alkyl esters were prepared from the corresponding substituted norketamines. Few of the latter have been reported since they have not been generally accessible via known routes. We report a new general route to many of these norketamines via the Neber (oxime to α-aminoketone) rearrangement of readily available substituted 2-phenycyclohexanones. We explored the use of the substituents Cl, Me, OMe, CF3, and OCF3, with a wide range of lipophilic and electronic properties, at all available benzene ring positions. The 2- and 3-substituted compounds were generally more active than 4-substituted compounds. The most generally acceptable substituent was Cl, while the powerful electron-withdrawing substituents CF3 and OCF3 provided fewer effective analogues.

Transcriptional changes in response to ketamine ester-analogs SN 35210 and SN 35563 in the rat brain.

BACKGROUND: Ketamine ester analogs, SN 35210 and SN 35563, demonstrate different pharmacological profiles to ketamine in animal models. Both confer hypnosis with predictably rapid offset yet, paradoxically, SN35563 induces a prolonged anti-nociceptive state. To explore underlying mechanisms, broad transcriptome changes were measured and compared across four relevant target regions of the rat brain. RESULTS: SN 35563 produced large-scale alteration of gene expression in the Basolateral Amygdala (BLA) and Paraventricular Nucleus of the Thalamus (PVT), in excess of 10x that induced by ketamine and SN 35210. A smaller and quantitatively similar number of gene changes were observed in the Insula (INS) and Nucleus Accumbens (ACB) for all three agents. In the BLA and PVT, SN 35563 caused enrichment for gene pathways related to the function and structure of glutamatergic synapses in respect to: release of neurotransmitter, configuration of postsynaptic AMPA receptors, and the underlying cytoskeletal scaffolding and alignment. CONCLUSION: The analgesic ketamine ester analog SN 35563 induces profound large-scale changes in gene expression in key pain-related brain regions reflecting its unique prolonged pharmacodynamic profile.