Electroencephalogram Studies of Induction and Recovery From Sevoflurane-Induced General Anesthesia
Phase: PHASE2
Conditions: Anesthesia
Sponsor: Massachusetts General Hospital

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Electroencephalogram Studies of Induction and Recovery from Sevoflurane-
Induced General Anesthesia 
 
NCT03503578 
02/24/2019 

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Electroencephalogram Studies of Induction and Recovery from Sevoflurane-
Induced General Anesthesia 
 
PRINCIPAL INVESTIGATOR: OLUWASEUN JOHNSON-AKEJU, M.D., M.M.SC 
 
I. BACKGROUND AND SIGNIFICANCE 
 
Sevoflurane is an anesthetic agent with a rapid induction, emergence and 
recovery profile.1 Evidence suggests that sevoflurane, similar to other ether derivatives 
in clinical use, exerts its physiological and behavioral effects by binding at multiple 
targets in the brain and spinal cord.2 Action at these targets includes potentiation of γ-
Aminobutyric acid (GABAA), glycine and two-pore potassium channels; and inhibition of 
voltage-gated potassium, N-methyl-D-aspartate (NMDA), muscarinic and nicotinic 
acetylcholine (ACH), serotonin, and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic 
acid (AMPA) channels.2-5 Despite detailed characterizations of the molecular and cellular 
pharmacology of anesthetics, the neural circuit-level mechanisms of general anesthesia-
induced unconsciousness are still being actively investigated.3,4 Extensive work has 
related in detail the electroencephalogram (EEG) patterns of propofol (2,6-di-
isopropylphenol) to its neural circuit mechanisms.6-12 Propofol primarily acts at GABAA 
receptors throughout the brain and spinal cord to enhance inhibition.3-5,13,14 It also 
potentiates glycine receptors, and provides inhibition to voltage-gated potassium, ACH, 
AMPA and Kainate channels.3-5 Given the diversity of receptor targets, a unitary 
hypothesis of the neural circuit mechanism underlying anesthesia-induced depression of 
consciousness does not seem likely. However, clinically, we have observed that 
sevoflurane induces stereotypical changes in the EEG that appear grossly similar to 
propofol and that may help relate this anesthetic vapor to its neural circuit mechanisms.  
Our previous work provides compelling evidence that unconsciousness under 
propofol is characterized on the EEG by alpha (8-12 Hz) oscillations that are coherent 
across the frontal cortex, delta (1-4 Hz) oscillations and high amplitude slow (0.1-1Hz) 
oscillations.7,9,11,13,15-17 Intracortical recordings during propofol-induced unconsciousness 
suggest that local and long-range cortical communication are impeded by temporally and 
spatially incoherent slow oscillations that exhibit phase-limited spiking.15 Analysis of the 
scalp EEG, a readily accessible measure of the average activity in large populations of 
cortical neurons, has established that propofol induces synchronous frontal alpha 
oscillations.7,9-11 Biophysical modeling provides further evidence that propofol induces 
coherent alpha activity by increasing GABAA conductance and decay time.6,12 This 
increase in GABAA conductance facilitates involvement of the thalamus in a highly 
coherent thalamocortical alpha oscillation loop.6,12 This pathologically coherent frontal 
alpha oscillation pattern reduces the dimensionality of the thalamocortical network, 
reducing the ability of the thalamus to project and coordinate exogenous inputs to the 
neocortex.12-14  
 
Coherent alpha oscillations have also been identified in animal studies of the 
inhaled anesthetics during unconsciousness.18-20 However, human studies examining 
this cortical dynamic are non-existent. Given that both sevoflurane and propofol are 
known to act at GABAA receptors,3-5,13,14 studying the EEG patterns elicited by 
sevoflurane using the novel paradigms we have previously established to study propofol 
will provide insights on the neural circuit mechanisms of sevoflurane. Previous studies 
employing EEG-based methods, including spectral analysis, bispectral analysis, auditory 
evoked potentials and methods for source localization, have been used to compare the 
conscious and unconscious states under general anesthesia, usually from surgical 
recordings taken before and after a rapid induction of general anesthesia.21 However, 
none of these methods have been used to track, on a moment-to-moment basis, the 

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transition to unconsciousness during a gradual induction of general anesthesia with an 
anesthetic vapor, nor the return of consciousness. 
 
We have recently studied the similarities and differences in EEG spectra and 
coherences between sevoflurane and propofol in data recorded during routine care of 
patients in the intraoperative setting [publication in review]. We found the following: (i) 
Similar to propofol-induced frontal alpha oscillations, sevoflurane is characterized by 
coherent alpha oscillations with similar maximum power and coherence occurring at 
~10-12 Hz; (ii) Also similar to propofol, sevoflurane is associated with slow oscillations 
characterized by similar power and coherence at frequencies < 1 Hz; (iii) In contrast to 
propofol, sevoflurane is associated with increased power in the theta band. These theta 
oscillations are coherent across the frontal cortex and may provide additional insight into 
the neural circuit mechanisms of sevoflurane especially with regards to thalamic 
dysrhythmia and deafferentation. However, since the EEG recordings we analyzed were 
obtained solely from frontal channels in a clinical setting, we were unable to perform 
detailed characterizations of changing behavior and consciousness during controlled 
induction and emergence, limiting our inferences to a clinically unconscious state. Also, 
the nature of data (frontal channel only) limited our ability to analyze the cortical 
dynamics underlying sevoflurane anesthesia-induced unconsciousness.  
 
Therefore, we propose a paradigm to use EEG measurements to study the 
dynamics of loss of the changes to well-designed sensory or cognitive “probes.” This 
study will help address the compelling need to replace the current clinical definitions of 
general anesthesia with neurophysiological and neuroanatomical definitions of the state 
of general anesthesia. By using principled auditory, and somatosensory asks, results 
from this study will help establish the relation between EEG changes and loss of 
consciousness. This may provide insights into the neural circuit mechanisms through 
which sevoflurane and other ether derivatives induce unconsciousness. The detailed 
characterization and comprehension of these mechanisms may prove vital for shaping 
future studies on establishing the neural correlates of consciousness.  
 
II. SPECIFIC AIMS 
The specific aims of this study are: 
AIM 1: To use high-density EEG recordings to characterize the different brain-states 
induced by the anesthetic vapor, sevoflurane. 
Hypothesis 1a. The onset and stable maintenance of sevoflurane-induced beta 
oscillations will signify a sedative brain state with a probability of response to auditory 
and nociceptive stimuli that approaches the awake state. 
Hypothesis 1b. The onset and stable maintenance of highly coherent sevoflurane-
induced frontal alpha and theta oscillations will signify a brain-state approaching a zero 
probability of response to auditory and nociceptive stimuli. 
Hypothesis 1c. Sevoflurane-induced large slow oscillations (at maximum amplitude) 
and burst suppression will signify a brain state with a zero probability of response to 
auditory stimuli. 
Hypothesis 1d. Return of consciousness will be preceded by return of gamma 
oscillations and the loss of large amplitude slow, theta, alpha and beta oscillations. 
 
AIM 2: To use high-density EEG recordings to understand the neural-circuit mechanism 
of action of ether anesthetics. 
Hypothesis 2a. The administration of an N-methyl-D-aspartate receptor antagonist 
(ketamine) during sevoflurane-induced unconsciousness (putative γ-aminobutyric acid 
mechanism) will antagonize and significantly diminish the amplitude of the alpha and 
slow oscillations.  

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Hypothesis 2b. The administration of an N-methyl-D-aspartate receptor antagonist 
(ketamine) during sevoflurane-induced unconsciousness (putative γ-aminobutyric acid 
mechanism) will produce globally non-coherent beta oscillations that are sufficient to 
maintain a brain state with a zero probability of response to auditory stimuli. 
Hypothesis 2c. The administration of an N-methyl-D-aspartate receptor antagonist 
(ketamine) during sevoflurane-induced unconsciousness (putative γ-aminobutyric acid 
mechanism) will antagonize the effects of sevoflurane on airway collapsibility by 
mediated by γ-aminobutyric acid inhibition on the hypoglossal motor neuron. 
 
AIM 3: To use inflammatory profiling of serum to understand the effect of sevoflurane on 
systemic inflammation  
Hypothesis 3a:  We hypothesize that sevoflurane-anesthesia will be associated with 
increased levels of pro-inflammatory cytokines interleukin 6 and TNF-alpha. 
Hypothesis 3b: We hypothesize that sevoflurane/ketamine-anesthesia will be 
associated with decreased levels of pro-inflammatory cytokines interleukin 6 and TNF-
alpha. 
 
III. SUBJECT SELECTION 
We will select 24 volunteer subjects (male and female) between the ages of 18-50 years.  
The subjects will be recruited using:                                                 
• 
An announcement of the study distributed through the Partners Public Affairs 
distribution list.                 
 
All study subjects will be American Society of Anesthesiologists (ASA) physical status 
classification P1. That is, all study subjects will be fit and healthy. A complete medical 
history will be taken and a complete physical examination will be given to rule out active 
and chronic medical problems.  
 
Primary Inclusion Criteria for Subjects 
• 
Between the ages of 18 and 50 
• 
Normal body weight and habitus, BMI ≤ 30 
• 
Non-smoker 
• 
American Society of Anesthesiologists (ASA) physical status classification P1 
 
Medical Reasons for Exclusion  
Chronic health conditions that will exclude subjects from the study include but are not 
limited to: 
 
Cardiovascular: 
hypertension, myocardial infarction, and coronary artery disease, 
peripheral vascular disease, arrhythmia, congestive heart failure, 
valvular disease 
 
Respiratory:  
sleep apnea, bronchitis, chronic obstructive pulmonary disease, 
smoking, shortness of breath 
 
Hepatic: 
hepatitis, jaundice, ascites 
 
Neurologic: 
seizure, stroke, positive neurologic findings on neurologic 
examination, multiple sclerosis, Meniere’s disease, Parkinson’s 
disease 

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Gastrointestinal: 
esophageal reflux, hiatal hernia, ulcer 
 
Endocrine: 
glucose intolerance, diabetes, thyroid disease 
 
Renal: 
acute or chronic severe renal insufficiency  
 
Hematologic: 
blood dyscrasias, anemia, coagulopathies, on anticoagulant 
therapy 
 
Musculoskeletal: 
prior surgery or trauma to head neck or face, arthritis, personal or 
family history of malignant hyperthermia 
 
Psychiatric: 
claustrophobia, history or treatment for an active psychiatric 
problem, depression 
 
Reproductive: 
pregnancy, breastfeeding 
 
Medications:  
regular use of prescription and non-prescription medications 
expected to affect CNS function 
 
Allergies:  
sevoflurane, phenylephrine, glycopyrrolate, ketamine, haldol 
 
 
Remuneration 
For successful completion of this protocol, subject remuneration will be $800. If the study 
subject is unable to complete the entire protocol, pro-rating of this remuneration will be 
as follows: 
• 
Study subjects who complete the medical screening evaluation but do not begin 
the anesthesia portion of the study will receive $50.  
• 
Study subjects who complete the medical screening evaluation, and one 
anesthesia study visit will receive $250. 
• 
Study subjects who complete all study portions will receive $800.  
 
Parking vouchers will be available on study days. 
 
IV. STUDY PROCEDURES  
Location:  
The screening visit will take place at the White 12 Clinical Research Center.  
 
The anesthesia portion of these studies will take place on White 5 at the Carl Rosow 
Center for Clinical Research. White 5 was constructed specifically for the performance of 
research on human subjects receiving the depressant medications used in anesthesia. 
The research area is built to the same standard as a typical surgical procedure room at 
MGH with respect to ventilation, temperature control, plumbing and electrical facilities, 
as well as storage for sterile supplies and linens. There is piped oxygen and air, wall 
suction and waste anesthetic gas removal. Electrical outlets are supported by the 
hospital’s backup generator. There is a fully-equipped anesthesia machine with an 
automated record keeper and standard anesthesia monitors. A fully equipped code cart 
is immediately available, and an emergency call system is connected to the White 3 
PACU. The code cart and medications are checked at intervals by personnel from the 

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OR pharmacy, and the anesthesia equipment is safety checked and maintained by 
Anesthesia Bioengineering. In short, research subjects receiving anesthetic drugs in this 
unit are given an identical standard of care as patients in our operating rooms. Any 
research protocol utilizing this space must first be reviewed by a DACCPM committee 
specifically created to ensure appropriate standards of anesthetic care and the safety of 
human subjects. 
 
Screening Visit:  
All subjects will complete a screening visit. Study subjects will arrive at the study site to 
undergo screening to ensure that they meet the basic inclusion/exclusion criteria for the 
study and give written informed consent. All questions will be explained as needed. All 
women of childbearing age will be tested for pregnancy. A urine hCG pregnancy test will 
be performed. If the test performed comes back negative for pregnancy, the subject will 
be enrolled. If the test performed comes back positive for pregnancy or the subject is 
breastfeeding, the subject will not be enrolled in the study. If the test performed comes 
back borderline, we perform another test.  
 
Consent Procedures: Prior to the study, each subject will sign an informed consent form. 
The investigator obtaining consent will explain in detail the protocol of the study, its 
purpose and potential benefits to the society. Subjects will be informed that if they feel 
uncomfortable with the study, they can choose to terminate the study at any time.  
 
Physical Examination: The subjects will be given a standard pre-anesthetic physical 
examination. Particular attention will be paid to the subject’s airway anatomy and 
neurologic function. Any abnormal findings on physical examination will be reason for 
exclusion from the study. Abnormal findings will be reported to the subjects and 
recommendations for medical follow-up will be given as needed. 
 
Screening Tests: A complete blood count, blood glucose level, liver function tests 
(LFTs), blood urea nitrogen (BUN), and creatinine (Cr) level will be obtained at the initial 
screening visit. For inclusion into the study protocol, each subject will be required to 
have a platelet count, blood glucose, BUN levels, Cr levels, white blood cell count, 
hemoglobin, differential and LFTs within 2 times the upper and lower limit of normal. Any 
subject who tests positive for any “street drugs” (amphetamine, barbiturates, 
benzodiazepines, cannabinoids, cocaine, opiates, or phencyclidine) may be excluded 
from the study. An ECG will also be performed.  
 
Randomization: Visits 1 and 2 will be randomized to determine their order of occurrence: 
sevoflurane only vs. sevoflurane with ketamine.  
 
Study Visit 1 and 2: 
Study subjects will arrive at the study site and confirmation of the study subjects’ fasting 
status (minimum 8 hours) will be made. A urine sample will be obtained for a toxic 
substance screen and for female study subjects, pregnancy testing. 
 
Blood Draws: For all subjects, we will acquire blood samples during Study Visits 1 and 2. 
The blood draws taken before and after anesthetic exposure will comprise the following: 
1.) approximately 2 ml of whole blood for analysis using the 44-analyte TruCultureMAP. 
Analytes in this map include interleukin 6 and TNF-alpha and, 2.) approximately 13 ml of 
blood will be acquired processed into serum and stored for downstream studies and 

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confirmatory assays of TruCultureMAP results.  
 
An EEG montage with a maximum of 256 channels will be placed on the subject. 
Standard 
physiological 
monitors 
for 
anesthesia 
will 
be 
placed 
including: 
electrocardiogram, pulse oximeter, and non-invasive blood-pressure cuff. End-tidal CO2 
measurements will be made by way of a capnogram attached to the expiratory port on 
the breathing circuit. Galvanic skin response will also be measured. A cuff pain delivery 
device will be attached to the gastrocnemius area of the lower leg. Baseline vital signs 
will then be taken. A peripheral intravenous line will be placed and an infusion of ringer’s 
lactate or normal saline will be started. Baseline physiologic measurements will be 
taken. 
 
The study protocol will be divided into 3 segments. These segments are defined in order 
as: Baseline Recording; Induction and Recovery from Hypnosis; Post-Anesthesia 
Recovery. 
 
One time before and one time after the study period, a calibrated cuff pain stimuli will be 
delivered to the gastrocnemius area of the lower leg using a validated cuff pain device 
titrated to 8-9/10 pain.22-25 A computer controlled air compressor (Hokanson Rapid Cuff 
Inflator) will inflate the cuff to a pre-specified pressure, and maintain the pressure at that 
level. Pressure will be maintained for up to 5-minutes to evaluate brain, cardiovascular, 
and subjective response to experimental pain. One advantage to using cuff algometry 
pain is that unlike more superficial methods (e.g. heat pain), cuff pain responses are 
unaffected by sensitization or desensitization of the skin, indicating that this procedure 
primarily assesses sensitivity in muscle and other deep tissues.22  
 
The following questionnaires will be administered during the baseline and recovery 
periods to assess pain intensity and quality, measure levels of emotional distress and 
cognitive state.:  
• 
PROMIS Numeric Rating Scale V1.0 – Pain Intensity 1A;  
• 
PROMIS Scale v2.0 – Neuropathic Pain Quality 5a;  
• 
PROMIS Scale v2.0 – Nociceptive Pain Quality 5a.  
• 
PROMIS Item Bank v.1.0 – Emotional Distress-Anxiety Short Form 8A (modified 
to ask for symptoms experienced within the past hour);  
• 
PROMIS Item Bank v.1.0 – Meaning and purpose Short Form 8A;  
• 
PROMIS Item Bank v.1.0 – Positive Affect Short Form 15A (modified to ask for 
symptoms experienced within the past hour); 
• 
The Clinician Administered Dissociative States Scale (CADSS) 
 
In addition, inattention, memory and risk assessment cognitive domains will be 
evaluated using the following three computer presented neurocognitive tasks 
respectively: 1) Global Precedence, 2) Operation Span and 3) Columbia Card tasks 
(https://www.neurobs.com).  
 
Recording of physiological measurements (blood pressure, heart rate, pulse oximetry, 
end tidal carbon dioxide, and galvanic skin response) will be initiated and maintained 
from the beginning of the study protocol until the subject is in the post-anesthesia 
recovery phase. 
 

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Induction and Recovery – Sevoflurane Only Protocol: Once the baseline measurements 
are completed volunteers will receive sevoflurane anesthesia, delivered via a Dräger 
Fabius Tiro (Telford, PA, USA) anesthesia machine, in high-flow oxygen/air admixture 
through a secured facemask to approximately achieve the following end tidal 
concentrations: 1, 2.1 and 3%. The inspired sevoflurane concentration will be adjusted 
until the desired end tidal sevoflurane concentration is achieved. Each sevoflurane end 
tidal concentration target will be maintained for approximately 8-12 minutes.  
 
 
Induction and Recovery – Sevoflurane and Ketamine Protocol: Once the baseline 
measurements are completed, volunteers will receive sevoflurane anesthesia, delivered 
via a Dräger Fabius Tiro (Telford, PA, USA) anesthesia machine, in high-flow oxygen/air 
admixture through a secured facemask to approximately achieve the following end tidal 
concentration: 2.1%. The inspired sevoflurane concentration will be adjusted until the 
desired end tidal sevoflurane concentration is achieved.  
 
After a stable target concentration of sevoflurane (approximately 1 MAC) has been 
established for approximately 12 minutes, a 0.75 mg/kg (lean body weight) bolus of 
ketamine will be administered. The sevoflurane concentration will be held constant for 
approximately 50 minutes.  

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The total time under sevoflurane will be approximately the same for both protocols.  
 
During the administration of sevoflurane the subject will be assigned to complete the 
following auditory task:  
 
Auditory task: Subjects will be asked to respond to auditory stimuli via button-press 
jittered at random between 4 and 8 second intervals in order to resolve the precise 
period at which loss and recovery of consciousness/responsiveness occurred at a fine 
temporal scale. The auditory stimulus will consist of two components, a train of “clicks” 
and the command “click the mouse”. Subjects will be asked to respond by pressing the 
mouse in the hand. The sound intensity for all stimuli will not exceed 85 dB sound 
pressure level (SPL), in accordance with National Institute for Occupational Safety and 
Health guidelines for occupational noise exposure (U.S. Department of Health and 
Human Services and National Institute for Occupational Safety and Health, 1998). 
 
Precautions: An experienced board certified anesthesiologist, who is not involved in data 
acquisition or other study functions will monitor and care for the subject according to the 
standards for delivery of anesthesia at this institution. The White 5 research unit is 
equipped with an anesthesia machine, and physiologic data are collected using a 
Metavision electronic anesthesia record. This research unit has been specifically 
designed and equipped according to MGH standards and Massachusetts regulations as 
an anesthetizing location, and it has been approved for this purpose by the Clinical 
Practices and Safety Committee of the MGH Department of Anesthesia, Critical Care 
and Pain Medicine. If more care is needed in the judgment of the supervising clinician, 
the subject may be transferred to the MGH White 3 PACU.  
 
Blood Pressure Management: Decreases in systemic arterial blood pressure are a 
known, expected side effect of sevoflurane. This is because sevoflurane depresses 
myocardial contractility and decreases systemic vascular resistance. This is manifested 
clinically as hypotension, defined as a decrease in mean arterial pressure (MAP) of 15%. 
Significant decreases in blood pressure caused by sevoflurane will be treated with 
intravenous phenylephrine. Phenylephrine is a synthetic sympathomimetic drug that 
selectively stimulates alpha-1 adrenergic receptors. In its clinical use, there is no 
appreciable effect on beta-adrenergic receptors. Resulting venoconstriction is greater 
than arterial constriction. Using an alpha-adrenergic agonist such as phenylephrine to 
treat hypotension is advantageous because this counters the sevoflurane-induced 
decrease in systemic vascular resistance. A particular advantage of phenylephrine is 
that it is usually administered by intravenous infusion and can therefore be easily titrated 
to effect.  
 
The phenylephrine infusion will be prepared according to usual practices of the MGH 
Department of Anesthesia and Critical Care: 1 ml of 1% phenylephrine (20 mg/ml) will be 
diluted in 250 ml normal saline or lactated ringer solution (final concentration 80 ug/ml). 
The 251 ml bag will be labeled using a standard, phenylephrine identification sticker 
confirming the contents, concentration and time and date of preparation of the 
phenylephrine mixture according to usual Department practices. The intravenous 
phenylephrine infusion will be titrated using a standard clinical infusion pump to maintain 
the mean arterial pressure within 15% of the study subject's baseline measurement. The 
infusion will be titrated and will be delivered within the range of 10 ug/min – 100 ug/min 
(15 ml/hr – 150 ml/hr). 

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Glycopyrrolate may also be used to treat changes in blood pressure. Glycopyrrolate is 
advantageous because this counters the decrease in heart rate without interfering with 
study measurements by crossing the blood brain barrier. Another particular advantage of 
glycopyrrolate is that it is usually administered by intravenous infusion and can therefore 
be easily titrated to effect. 
 
Delirium Management: We have carefully selected ketamine 0.75mg/kg based on 
evidence that this dose is safe when used alone or in combination with sevoflurane. This 
dose is within the range that is typically administered in the MGH operating rooms as an 
anesthetic adjunct to help limit the exposure to narcotics. This dose has also been used 
in clinical research studies of depression at MGH (0.75mg/hr over 45 minutes; N-methyl-
D-aspartate Antagonist (Ketamine). Thus, the administration of an adjunctive dose of 
ketamine as a bolus, with emergence from anesthesia approximately 60 minutes 
afterwards should constitute minimal delirium risk. However, in the event the subject is 
not oriented to space and place, attempting to pull out intravenous lines, is adjudged to 
constitute a risk to self and/or study staff, haloperidol 1-2 mg will be administered by one 
of the study anesthesiologists. 
 
Airway Management: Induction of general anesthesia will cause predictable changes in 
respiratory function. General anesthetic agents, including sevoflurane, can produce 
apnea after the loss of consciousness. While the study subject is awake, oxygenation 
and ventilation will be spontaneous. The standard clinical approach for airway 
management in surgical patients is for the anesthesiologist to assist oxygenation and 
ventilation after the loss of consciousness. With progressive loss of consciousness, the 
study subject will hypoventilate and become apneic. The staff anesthesiologist will use 
standard airway maneuvers to maintain normal oxygenation and ventilation. These 
maneuvers include a jaw and chin thrust and assisted ventilation with a mask and bag 
as part of a circle circuit. Ventilation will be manually assisted as needed. At all times, 
the inspired oxygen concentration and expired carbon dioxide waveform and partial 
pressure will be monitored continuously. The minimum inspired oxygen concentration 
will be 30%. Thus, an anesthesiologist may manually ventilate the subject. The end-tidal 
(capnogram) carbon dioxide levels will be maintained within 10% of the baseline values. 
Manually assisted oxygenation and ventilation will continue until the subject recovers 
spontaneous ventilation.  
 
Post-Anesthesia Recovery: Following Induction and Recovery, the subject will be 
monitored for post-anesthesia recovery for up to 1 to 2 hours as is done for patients 
following ambulatory surgery. The subject will be discharged based on established MGH 
Department of Anesthesia practices for discharge from the hospital following ambulatory 
surgery. The subject must have stable vital signs, i.e., within 20% of pre-study values, be 
able to respond appropriately to normal commands, be pain-free, be free from any 
nausea and vomiting, and have no bleeding from the intravenous site. Subjects must be 
able to walk unassisted and have an accompanying adult to escort them home. Subjects 
will be advised not to return to work for 12 hours and be advised against driving or 
operating heavy equipment for 24 hours.  
 
VI. BIOSTATISTICAL ANALYSIS  
We are using an endpoint of performance score on the Global Precedence task to guide 
sample size determination. Our null hypothesis is the average within subject difference 
in performance score for sevoflurane compared to sevoflurane/ketamine is zero. Our 

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alternative hypothesis is the average within subject difference in performance score for 
sevoflurane compared to sevoflurane/ketamine is 3. If we assume that the variance in 
performance score is 9, a type I error of 0.05 and a minimum power of 0.90 for a one-
sided z-test, the sample size determinations for our study is 9. Therefore, we should 
detect a clinically relevant improvement in performance score if we successfully enroll 
only 9 subjects. To account for data unanticipated events that may lead to poor data 
quality or data loss, we are proposing to enroll 12 subjects. 
 
EEG acquisition Preprocessing: Continuous EEG will be recorded with up to 256 
channels. All data will be stored for subsequent off-line analysis. We will apply an anti-
aliasing filter and down-sampled the EEG data to 500 or 250 Hz before analysis. 
Bandpass filtering of the acquired signal will vary depending upon the features of the 
EEG data of interest. EEG signals will be remontaged to a nearest-neighbor Laplacian 
reference, using distances along the scalp surface to weigh neighboring electrode 
contributions. Trigger signals will be sent from the stimulus delivery system to the EEG 
recording system to tag the stimulus events for subsequent binning, temporal epoching 
and averaging of EEG data into the relevant evoked potential averages according to 
stimulus type and experimental condition.  
 
Video recording to assess level of arousal: Video of the study subject may be recorded 
during the baseline and induction and recovery from hypnosis periods to assess the 
study subject’s level of arousal. In particular, from clinical experience and previous 
studies, we note that subjects will exhibit varying levels of arousal after the loss of 
consciousness, visible in terms of spontaneous movement, which can be recorded on 
video and then correlated with the EEG. Permission to record video for these data 
analysis purposes will be obtained from the main research consent form. The video 
recordings will be stored in a locked file in the investigators office.  
 
Behavioral Analysis: The probability of response to the click and verbal stimuli and the 
difference in probability of response will be estimated by using Bayesian Monte Carlo 
methods to fit a state-space model to these data. To perform group-level analyses, we 
will align the behavioral data across subjects with respect to each subject’s LOC time for 
induction and with respect to each subject’s ROC time for emergence. We will then pool 
the responses within bins. The pooled data will be used to estimate group-level 
probabilities of response using the state-space model. 
 
Spectral Analysis: We will compute spectrograms for all subjects using the multitaper 
method. We will also compute group-level baseline-normalized spectrograms for 
induction and emergence by taking the median across subjects with the data aligned to 
the LOC and ROC time points, respectively. To determine if the group-level spectrogram 
is significantly greater than baseline, we will perform a two-group-test as implemented in 
chronux. 
 
Eigenvalue and Modal Projection Analyses: We will perform an eigenvector 
decomposition analysis of the cross-spectral matrix to identify the principal modes of 
oscillation in the conscious and unconscious states and to analyze how activity within 
these principal modes change through time. We will estimate the cross-spectral matrices 
Pbaseline(f) and Punconscious(f) at each frequency f using the multitaper method using data 
from the full baseline period, and segments of at least 5 min extending from LOC to 
ROC, respectively, for each subject. An eigenvalue decomposition of the median cross-
spectral matrix at each frequency will be performed. Each eigenvector describes a 

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coherent spatial distribution or mode of oscillation, and the corresponding eigenvalue 
quantifies the power in this mode. We then use modal projection analysis to characterize 
how power within principal modes change as a function of time. A permutation-based 
procedure will be performed to assess statistical significance for the modal projection 
analysis.  
 
Phase–Amplitude Modulation Analysis. We will analyze the relationship between low-
frequency phase (0.1–1 Hz) and alpha/beta (8–14 Hz) amplitude by calculating a phase–
amplitude histogram. Bandpass filters will be applied to construct narrow-band slow and 
alpha/beta signals. We will then apply the Hilbert transform to each signal and compute 
the low-frequency oscillation phase and alpha oscillation amplitude. To construct the 
histogram, we will assign each temporal sample to one of 18 equally spaced phase bins 
based on the value of low-frequency oscillation phase, averaging over 2-min epochs. 
The histogram in each phase bin is the average of alpha oscillation amplitude for all 
samples within the bin, normalized by the average of alpha oscillation amplitude over the 
entire 2-min epoch. To assess statistical significance for the histogram, a permutation 
test will be performed. 
 
Anticipated Findings: Changes in EEG recordings to auditory, somatosensory, may aid 
principled approaches to brain state monitoring and targeting during general anesthesia.  
 
VII. RISKS AND DISCOMFORTS 
Sevoflurane risks: The risks involved in the administration of sevoflurane include, 
nausea, vomiting and coughing. Other possible side effects of sevoflurane are increased 
salivation, fever, and tingling in the extremities or hepatic injury. 5% to 9% of patients 
experience changes in heart rhythm, agitation, difficulty breathing, decreases in blood 
pressure, shivering and tiredness. Study subject hemodynamic parameters will be 
continuously monitored to ensure that appropriate medical intervention will be instituted 
for any clinically significant hypotensive or bradycardic episodes. 
 
Phenylephrine risks: Risks involved in the administration of phenylephrine include 
hypertension and bradycardia.  
 
Glycopyrrolate risks: Risks involved in the administration of glycopyrrolate include 
hypertension and tachycardia. 
 
Ketamine risks: Possible side effects reported during ketamine infusion include an 
increase in heart rate and blood pressure, arrhythmia, increased salivation, increased 
bronchial secretions, horizontal nystagmus, euphoria, and hallucinations. Any acute 
changes in physiology, such as changes in heart rate or blood pressure, will be treated 
using standard anesthesiology practices. Rare side effects are allergic reactions (skin 
rash), pain at the site of injection, increased intraocular pressure, ulcerations and 
inflammation in the bladder (reported in ketamine abusers). 
 
EEG risks: Electrodes placed on the scalp may cause temporary redness. 
 
 
Risks 
of 
Intravenous 
Line 
(IV): 
Risks 
involved 
include 
bruising, 
infection, 
lightheadedness, and fainting.  
 

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Hearing risks: Presented auditory stimuli will not exceed 85 dB SPL, as recommended 
by the National Institute for Occupational Safety and Health, and present minimal risk of 
hearing loss (U.S.Department of Health and Human Services and National Institute for 
Occupational Safety and Health, 1998). 
 
Risks associated with cuff pain stimuli: Risks of cuff pain include mild transient bruising 
associated with inflation of the cuff. In our experience, this is infrequent (< 5 % of 
cases).In fact, cuff pain has been well tolerated by both healthy adults and chronic pain 
patients, and according to our data, the pain intensity has been found to remain within 
stable limits for the duration of the scan run (e.g. 1st 2min: 35.9±12.4; 2nd 2min: 
42.9±14.0; final 2min: 43.4±21.6, on a scale of 0-100). Our previous experience has also 
demonstrated that pain ratings go down to nil within several seconds of stimulus 
termination. A simple button press can rapidly deflate the cuff, ensuring subject safety. 
 
VIII. POTENTIAL BENEFITS 
There are no direct benefits to the individual subjects involved in this study. Sevoflurane- 
and/or sevoflurane plus ketamine-induced EEG dynamics may aid principled 
approaches to brain state monitoring and targeting during general anesthesia. They may 
also lend insights into anti-nociceptive or pain mechanisms.  
 
IX. MONITORING AND QUALITY ASSURANCE 
No identifiers other than study ID’s will be included in the dataset. EEG data will be 
stored for later off-line analysis. Safety monitoring will include immediately available 
cardiopulmonary resuscitation cart, oxygen, ambu bag and defibrillator for use in need of 
an emergency. A fully functional O2 delivery system monitors, and a fully stocked 
anesthetic cart with equipment for airway management will also be available.  
 
One clinical board certified level anesthesiologist who is not taking part in the execution 
of the study protocol will be present at all times and responsible only for the medical 
management of the patient. This anesthesiologist will have the ability to halt the study at 
any point if he/she determines that this would be in the best interest of the subject. Prior 
to the start of the study it will be verbally reiterated what each person is responsible to 
execute if an emergency should occur as noted: 
 
1. Clinical Anesthesiologist – will call and run code  
2. Study Anesthesiologist – will assist with code, call White 3 PACU if needed 
3. Clinical Coordinator – Keep time and notes of all events 
 
Study Stopping Criteria: 
Prior to study initiation, the subject will be instructed to alert the study staff to the 
occurrence of pain, discomfort or anxiety. The study physician will interrupt the study 
and attempt to alleviate the source of pain, discomfort, or anxiety.  
 
To eliminate any ambiguity with hand signals, the subject will be instructed to give a 
“thumbs up” for an affirmative answer, and to wiggle his/ her index finger back and forth 
to give a negative answer. No more than 2 separate incidents will be allowed before the 
study is aborted. For any single incident, the investigators will spend no more than 20 
minutes to alleviate pain or discomfort before the study is aborted. At any time prior to 
the 20-minute or 2-alert limits, the clinical anesthesiologist can require the study 
anesthesiologist to abort the study if, in his or her judgment, the safety of the subject is 

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in question. Also, the clinical anesthesiologist can also require the study anesthesiologist 
to abort the study at any time for any subject safety-related concerns. 
 

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