Clinical trial NCT03435055
Conditions: Healthy
Sponsor: University of Michigan
Start date: 2017-07-21; Completion date: 2019-10-11
Enrollment: 21 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to understand how a commonly used drug, nitrous oxide, acts on the brain to reduce pain. Nitrous oxide is commonly used in anesthesiology but there is limited knowledge on how this drug affects functional networks in the brain.

Detailed description:
The objective of this study is to identify the network transformations that account for the analgesic effects of nitrous oxide. Our hypothesis is that analgesic doses of nitrous oxide increase network efficiency and disrupt normal pain processingOur approach is to administer subanesthetic nitrous oxide during the acquisition of fMRI (functional magnetic resonance imaging) and EEG (electroencephalogram).

Arms: arm 1: Each volunteer will participate in one scanning visit in which simultaneous functional magnetic resonance imaging (fMRI) and electroencephalogram (EEG) data will be collected wherein they receive placebo (20 minutes) followed by inhaled nitrous oxide at subanalgesic levels (35% inhaled concentration) over 40 minutes.
Interventions: intervention 1: Nitrous Oxide Gas for Inhalation
Intervention descriptions: intervention 1: Each volunteer will participate in one scanning visit in which simultaneous functional magnetic resonance imaging (fMRI) and electroencephalogram (EEG) data will be collected wherein they receive placebo (20 minutes) followed by inhaled nitrous oxide at subanesthetic levels (35% inhaled concentration) over 40 minutes.

Protocol documents:
Prot_SAP_000.pdf: 
Network-Level Effects of Nitrous Oxide in the Human Brain 
NCT: 03435055 
Document Date: 07/23/2020 
 
 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
Network-Level Effects of Nitrous Oxide in the Human Brain 
 
BACKGROUND  
General anesthetics can, in coarse terms, be mechanistically classified as “GABAergic” and “non-
GABAergic.” Clearly there is great diversity among GABAergic anesthetics such as propofol and the 
halogenated ethers. However, in general these drugs tend to be potent hypnotics and weak analgesics 
that are associated with a slow-wave EEG pattern at concentrations consistent with surgical anesthesia. 
For nitrous oxide, the GABA receptor is not the primary therapeutic target;1-4  this drug is a relatively 
weak hypnotic and potent analgesic that activates higher-frequency activity of the EEG. Despite these 
differences, nitrous oxide is pharmacologically classified and clinically used as general anesthetics, 
alongside propofol and the halogenated ethers. The study of anesthetic mechanisms will simply not be 
complete without a neuroscientific understanding of these drugs and the use of fMRI and EEG is the 
best technology available for systematic human studies. Identifying common neural principles of the 
anesthetic action of nitrous oxide and GABAergic drugs would be a major advance for the field of 
anesthesiology that would, for the first time, establish a common and empirically-demonstrated 
neurobiology of anesthetic-induced unconsciousness. This has not been possible with prior molecular or 
neurophysiological approaches, which motivates our focus on network-level properties.  
Of further clinical and scientific interest, nitrous oxide disrupts pain processing at subanesthetic doses. 
Over the past two decades there has been increased excitement about techniques that probe the 
functional,5 structural,6 and molecular7 mechanisms involved in human pain and its amelioration. 
However, less attention has been 
focused on how brain networks mediate 
pain and, moreover, how these same 
networks are modulated by centrally-
acting analgesics such as nitrous oxide. 
We propose that the dose-dependent 
analgesic and anesthetic actions of 
these drugs can be understood as 
emergent network properties. The 
hypotheses motivating the currently 
proposed studies are summarized in the 
figure on the right.  
Data derived from chronic centralized pain states suggest that pain occurs when particular brain regions 
functionally connect with other networks of the brain. Our recent work in fibromyalgia patients5,8 has 
found that the insula, a key higher-order sensory structure, is hyperconnected to the brain’s Default 
Mode Network (DMN), a constellation of brain regions involved in self-referential thinking (when an 
individual is not actively engaged with their external environment). Importantly, this enhanced 
connectivity is associated with self-reports of clinical pain intensity, and successful pharmacologic 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
treatment is associated with alternative network connectivity patterns (see preliminary data).9 
Therefore, like consciousness and anesthesia, pain and analgesia can be understood by examining brain 
networks and their alterations. 
Relevant Literature: The relevant literature for this study pertains to the brain mechanisms for both 
analgesic and anesthetic doses of nitrous oxide.  
There is one neuroimaging study that address the analgesic role of nitrous oxide (Gyulai et al20). In this 
study, the authors sought to identify the regions of the brain associated with the antinociceptive effects 
of nitrous oxide to a noxious heat stimulus. Measuring cerebral blood flow, Gyulai et al found that heat 
stimulus increased cerebral activation of the thalmus, anterior cingulate and supplementary motor area. 
Although nitrous oxide increased activation of subregions of the prefrontal cortex, nitrous oxide 
administered while the heat stimulus was applied reduced activation in the thalamus and 
supplementary motor area. As this study speaks to the analgesic effects of nitrous oxide, it does not 
measure the brain network properties of nitrous oxide administration at the anesthetic dose.  
Preliminary Studies: We have shown previously that fibromyalgia patients display increased resting state 
connectivity between pain regions such as the insula and other non-pain related areas such as the 
DMN.5,8 Moreover, reductions in this connectivity were associated with reductions in clinical pain: the 
degree of this connectivity was directly correlated with clinical pain ratings. In our Anesthesiology 
article,9 we examined the effect of pregabalin, an efficacious analgesic, on chronic clinical pain in 
fibromyalgia. Prior to and following each treatment period (drug/placebo), patients underwent fcMRI of 
the entire brain during rest. We placed a seed voxel within the posterior insula, a region where we 
previously observed elevated glutamate in chronic pain patients,7 and we assessed connectivity of this 
structure to the DMN before and after pregabalin and placebo. Interestingly, we found that during 
pregabalin treatment, but not placebo, patients displayed decreased insula connectivity to the inferior 
parietal lobule (IPL; a DMN region), which was paralleled by reductions in clinical pain: patients with 
greater reductions in insula-DMN connectivity displayed greater analgesia. These findings are relevant to 
the proposed study because 
they demonstrate that we 
can measure changes in 
brain networks during 
pharmacologic treatment 
and these changes are 
related to analgesic drug 
action.  
Figure above shows that 
change in connectivity between the posterior insula and IPL correlates with change in spontaneous 
clinical pain. We acknowledge that network perturbation by nitrous oxide may involve other brain 
regions and networks.  
 
 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
OBJECTIVE 
The objective of this study is to identify the network transformations that account for the dose-
dependent analgesic and sedative-hypnotic effects of the non-GABAergic anesthetic, nitrous oxide. Our 
hypothesis is that analgesic doses of nitrous oxide increase network efficiency and disrupt normal pain 
processing, while higher doses significantly reduce efficiency and disrupt normal conscious processing. 
Our approach is to administer two dosing levels of nitrous oxide during the acquisition of fMRI and EEG. 
To differentiate analgesic action of nitrous oxide from other processes, we will compare changes in brain 
response to noxious pressure stimuli. Our approach is innovative in that no study to date has acquired 
fMRI or EEG data to assess non-GABAergic anesthetics or to assess the relationship between analgesia 
and anesthesia. The rationale for this study is that, once completed, we will have established a detailed 
understanding of the therapeutic effects (analgesia and anesthesia) of a unique and understudied class 
of anesthetic drugs. Our expectation is that the knowledge derived from this study will have a positive 
impact by filling in a major gap in our understanding of anesthetic mechanisms and will potentially 
facilitate a generalized framework for anesthetic-induced unconsciousness. Furthermore, the study will 
provide a sophisticated network-level understanding of the relationship between the neural states of 
analgesia and anesthesia. 
SPECIFIC AIM 
1. To identify dose-dependent changes in brain networks using fMRI studies in healthy volunteers 
receiving nitrous oxide.  
2. To identify dose dependent changes in brain feedback connectivity using EEG in healthy volunteers 
receiving nitrous oxide.  
METHODOLOGY 
Here we describe a mechanistic study in healthy human volunteers to achieve the objectives of our 
specific aims. 
Participant Recruitment: We will recruit 25 individuals for an fMRI/EEG study.  
Healthy volunteers will be identified through the UM Clinical Studies website 
(http://www.umclinicalstudies.org) for research volunteers. We will also utilize the Minority Health 
Research Program (MHRP) to help recruit minorities into our study. Our goal is to recruit groups of 
subjects that are representative of the southeastern Michigan population. (See Appendix A for ethnic 
and racial targeted enrollment). All interested volunteers will first contact Dr. Mashour or the study 
coordinator for a preliminary screen to determine if the subject meets the inclusion/exclusion criteria 
and that the subject understands the research study. Specifically, the screening script will be read to 
potential participants, after which, they will be screened over the phone for eligibility using phone 
screening questionnaires. All screening documents have been attached to the Subject Recruitment 
section of the IRB application. Dr. Mashour or the study coordinator will also answer any questions that 
the potential participants may have at this time. If the potential participant responds to the psychosis, 
the mood disorder, or the Patient Health Questionnaire-9 (PHQ-9) depression screening questionnaire in 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
a manner that would potentially indicate a depressive/mood diagnosis, he/she will be referred to the 
Depression Center at the University of Michigan Health System (1-800-525-5188). Eligible participants 
will then be scheduled to participate. Dr. Mashour and the study coordinator are responsible for 
obtaining full written informed consent on the day of the first study visit upon the participant's arrival. 
Privacy will be maintained throughout the screening process per normal UMHS HIPAA privacy policies. 
Study participants:  
Inclusion criteria include: age 21-40, right-handed, American Society of Anesthesiologists Class 1 physical 
status, body mass index <30, with Mallampati 1 or 2 airways.  
Exclusion criteria include: history of obstructive sleep apnea, history of difficult airway with previous 
anesthetics, gastroesophageal reflux, hypertension or other cardiovascular abnormalities, pulmonary 
hypertension, history of drug use, history of chronic alcohol abuse, history of pain disorders, history of 
depression, psychosis or bipolar disorder, history of seizures or other neurologic disorders, history of 
methylenetetrahydrofolate reductase deficiency (a contraindication for nitrous oxide), history of known 
hypersensitivity to midazolam, Zofran, labetalol or glycopyrrolate, pregnant and / or nursing mothers, 
contraindications to the neuroimaging methods used (i.e. metal implants), tattoos in the head/neck 
region (all other tattoos may be subject to study exclusion by investigators), and any impairment, 
activity, or situation – as determined by the study coordinator or primary investigators – that would 
prevent satisfactory completion of the study protocol. 
Power analysis: We calculated power using data from fMRI analyses. Based on an effect size (Cohen’s d) 
derived from preliminary data, 22 participants are required for a 90% power to detect a statistically 
significant reduction in average path length (reflecting network efficiency).  In case of drop out across, 
we will continue to recruit until we have 22 volunteers with completed data sets.  
EXPERIMENTAL PROTOCOL 
Each participant will participate in two study visits, one consent/pre-scan visit and one scanning visit 
within 3 days apart. During the consent/pre-scan visit, participants will undergo consent for 
participation in the study and Quantitative Sensory Testing (QST). During the scanning visit, 
simultaneous fMRI/EEG data will be acquired while participating in additional tasks and administration 
of nitrous oxide gas.  
Consent/Pre-Scan Visit 
The study coordinator will determine whether the volunteer meets inclusion and does not meet 
exclusion criteria prior to consent. Following consent, each volunteer will complete a resting state 
questionnaire and QST familiarization/training during EEG acquisition. MR-compatible EEG cap and 
electrodes will be affixed to the participant’s scalp to monitor brain activity during QST training. QST will 
consist of a constant pressure applied to the lower leg and/or visual stimulus outside of the scanner. 
Following each stimulus, participants will rate the pain intensity (0 = “no pain, 100 = “worst pain 
imaginable”) and pain unpleasantness (0 = “neutral”, 100 = “extremely unpleasant”). Participants will be 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
asked to complete another resting state questionnaire following training on simultaneous visual 
stimulus and pressure cuff. During this visit, participants will also be seen briefly by an anesthesiologist 
for an airway assessment to check for eligible Mallampati 1 or 2 airways.  
Scanning Visit 
Simultaneous Functional Magnetic Resonance Imaging/Electroencephalogram (fMRI/EEG) 
Each volunteer will participate in one scanning visit in which simultaneous fMRI/EEG data will be 
collected wherein they receive placebo (20 minutes) followed by inhaled nitrous oxide at analgesic 
levels (35% inhaled concentration) over 40 minutes, then a nitrous oxide dose consistent with 
anesthetic-induced unconsciousness (70%) for 15 minutes [See Figure 1]; 
 
Figure 1. Study design schematic 
Upon arrival, participants will complete baseline questionnaires (Part 1a). Prior to drug exposure, 
volunteers will be given an altered states of consciousness (ASC) questionnaire followed by a baseline 
resting state fMRI scan. During the placebo period, fMRI data will be acquired during additional tasks: 
visual stimulation task, evoked pressure pain applied to the lower leg induced by a cuff/automated 
tourniquet, and a combination visual stimulation and pressure pain cuff task. These scans will be 
repeated during both analgesic and anesthetic doses of nitrous oxide. End-tidal nitrous oxide 
concentrations will be assessed by standard gas analyzers during spontaneous ventilation through a 
secured face mask with tight seal.  
All subjects will have MR-compatible EEG electrodes affixed to their scalp for monitoring during the 
study. Participants will first complete an altered states of consciousness questionnaire. Continuous EEG 
will be acquired during a baseline (rest) period followed by three additional tasks mentioned previously. 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
For each participant, these tasks will be repeated at a low dose of nitrous oxide that is normally 
associated with analgesia (35% inhaled concentration) and a higher dose of nitrous oxide that is 
associated with anesthesia. Following administration of the analgesic dose of nitrous oxide, participants 
will be asked to complete a questionnaire to assess altered states of consciousness. Each testing session 
will conclude with a 15-minute continuous EEG acquired during the participant’s recovery of 
consciousness. Following testing, the participants will be asked to complete an altered state of 
consciousness questionnaire.  
To maximize safety, nitrous oxide will be delivered using MRI-compatible anesthesia machines at the 
University of Michigan hospital, and anesthetic doses of nitrous oxide will be administered outside of 
the scanner, where airway patency and physiological stability will be established prior to imaging the 
state of anesthetic-induced unconsciousness. At least two fully-trained anesthesiologists will direct all 
anesthetic administration. In order to maximize clinical monitoring during emergence, recovery from 
anesthetic doses will occur outside of the MRI scanner as well. All participants will receive ondansetron 
(4mg IV) with an additional dose of dexamethasone (4mg IV) if needed to prevent nausea and vomiting. 
In addition, glycoyrrolate (0.42-0.4 mg IV), labetalol (5-10 mg/kg IV), and midazolam (1-2 mg IV) will be 
available for use to mitigate any side effects, as needed.  Also, standard intraoperative monitors 
(electrocardiogram, pulse oximetry, capnography) will be used.  
Subjects will only be discharged if standard clinical criteria for discharge at UMHS is met. This includes 
being awake, fully responsive, able to walk, able to urinate, and no significant nausea and vomiting. The 
medication will be given as needed during the recovery period while being monitored by Dr. Mashour or 
another fully trained anesthesiologist. Participants will not be allowed to drive home and transportation 
will be provided. In addition, approximately 24 hours after the nitrous anesthetic, the study coordinator 
will call the participant to follow-up and ensure that there are no adverse events. If an adverse event is 
detected, the study coordinator will immediately contact Dr. George Mashour and determine the 
appropriate course of actions to be taken. 
DATA COLLECTION 
Resting State Functional Connectivity (fcMRI).  
During fcMRI, participants will either be at rest or receiving a pain stimulus via a pressure cuff applied to 
the left lower leg evoking tonic pressure with pain of 40 on a 100-point numerical rating scale (See 
below; 0=no pain; 100=worst pain imaginable). Functional connectivity fMRI will be performed on a 3 
Tesla Philips Achieva (Best, Netherlands) using a T2* weighted echo-planar sequence with the following 
parameters: TR/TE = 2000/30ms, flip angle=90˚, FoV = 200x200mm, 48 AC-PC aligned slices, slice 
thickness 3 mm, scan time = 6min. These data will be analyzed using two approaches: a model-free 
independent component analysis (ICA) and seed-voxel based method (from evoked pain fMRI; See 
below).  
 
 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
Evoked pressure pain with blood pressure cuff: 
Calibrated cuff pain stimuli will be delivered to the gastrocnemius area of the dominant leg using a 
validated cuff pain device that has been recently adapted to the MRI scanning environment. 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. Subjects will participate in one test session of cuff pain prior to 
the EEG and the fMRI session. Pressure pain will be applied for a continuous 6-minute period three 
times during the fMRI protocol to evaluate functional brain connectivity response to deep pain (see 
below). 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. We have considerable 
experience applying these techniques in both healthy adults and chronic functional pain patients, and 
have found that all subjects are able to tolerate these procedures without any lasting discomfort.  
Training Session. A training session with the cuff will be used to familiarize subjects with the stimuli and 
rating procedures and determine appropriate stimulus intensities to be used subsequently in the 
imaging session (see below). This can take place on the day of fMRI or during the in-clinic consent visit. 
During training, subjects will be maintained in a seated position in a chair with the left foot resting on a 
support at a slightly elevated position (but at a lower level than that of the hips, in order to facilitate 
blood circulation in the leg).  
Testing will begin with an ascending series of pressure stimuli starting at 60 mmHg and increasing 20 
mmHg increments. Ten seconds after the end of each stimulus, subjects will complete two 0-100 
numerical rating scales: pain intensity (0 = “no pain”, 100 = “worst pain imaginable/extreme pain”) and 
pain unpleasantness (0 = “neutral”, 100 = “extremely unpleasant”). The ascending series will end when a 
pain intensity rating of >70/100 is obtained. Participants may also receive a continuous tonic pressure 
for 6 minutes to their lower leg using the cuff, and asked to rate the intensity and unpleasantness of the 
stimulus at certain intervals during the test, up to three times. A short break will be given between each 
pressure test.  
This training session will have the effect of rendering subjects non-naïve to the experimental conditions 
in the imaging session, an aspect that might be argued to have some impact on the imaging results 
(particularly with regard to brain activity underlying cognitive and emotional functions). However, the 
training session has several advantages that outweigh these concerns: (1) identification and exclusion of 
individuals with unstable ratings, (2) a thorough training in the use of the rating scales, and (3) the 
reduced potential for developing experiment-related anxiety and head motion in the imaging session.  
Imaging session. On the day of the imaging session, the Pain0-Pain70 pressures will be briefly 
recalibrated prior to scanning using procedures similar to those used during the training session.  
During the fMRI scan runs, subjects will receive cuff pressures calibrated to each subject’s Pain30-50 
applied for a continuous 6-min period to evaluate functional brain connectivity response to tonic deep 
pain. Following each cuff pain run, participants will be asked to rate the degree of pain intensity and 
unpleasantness on a 100-point numerical rating scale. 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
Visual Stimulation Task 
During the visual stimulation, participations will be undergoing a 3-minute period of dynamic visual 
stimulation tasks which consists of alternating 20 second blocks of flashing (8 Hz frequency) blue-yellow 
annulus checkerboard and a static fixation cross. The checkboard will be presented at a fixed 
illumination level in within the range of 4.5 to 76 lux. Following stimulation, participants will be asked to 
rate the intensity and unpleasantness of the stimulus on a 100-point numerical rating scale.  
Electroencephalogram (EEG) 
All subjects will have EEG electrodes affixed to their scalp for monitoring during the study. We will use 
the Brain Products MR-compatible EEG system for EEG, as it offers the capability to record 64 channels 
of EEG data in the MR environment. This equipment has been tested and approved for safe use by the 
University of Michigan Biomedical Engineering Department.  
The EEG electrodes and the cap to hold the electrodes will be disinfected after each use with an FDA-
approved solution provided by EGI Geodesic System (there is no standard UMHS protocol for this). The 
EEG electrodes have minimal risk but may cause a slight irritation to the skin that will resolve on its own. 
Acquisition of EEG data will occur during both the consent/QST visit and the fMRI scanner visit.  During 
the QST visit, EEG data will be acquired during rest periods and stimulus presentation. During the 
scanner visit, EEG will be acquired simultaneous with the fMRI data. The sampling frequency at which 
data will be acquired is 5000 Hz and EEG data will undergo pre-processing prior to assessment of brain 
network dynamics. 
Questionnaire 
Each participant will complete a series of questionnaires to further characterize the study participants 
on the day of testing prior, during and after the experiment. Before testing, participants will complete 
general psychometric surveys to assess personality, emotion (positive and negative affect), depression, 
anxiety and handedness21-27. All surveys have been attached to the Survey Research section of the IRB 
application. 
During the nitrous oxide protocol, the participants will complete four additional questionnaire 
components; an altered states of consciousness and resting state questionnaires, as well as intensity 
and unpleasantness rating. The altered state consciousness (ASC) questionnaire is a survey completed 
by the participant on a visual analog scale (0 – 10) to assess the affective experience of nitrous oxide 
administration during the study. The resting state questionnaire assess resting state cognition during 
non-sensory experiences (resting state scan), such as mind-wandering and self-referential thinking. 
Intensity and unpleasantness rating will mimic the quantitative sensory testing session, given prior to 
each experiment, where participants are asked to rate the intensity and unpleasantness of the sensory 
stimuli on the Gracely Box Scale, previously used in our lab.  
The ASC questionnaire should take 10 minutes to complete and will be administered at baseline, and 
after the analgesic and anesthetic dose periods. The resting state questionnaire will be administered 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
following the resting state periods and stimulus periods during the consent/QST visit. Intensity and 
Unpleasantness ratings will be acquired during and following both resting scans and sensory stimuli 
scans (cuff pain and visual stimulation). 
The final survey should take less than one hour, may be completed online and should be completed 
within 48 hours after the study visit. This survey will contain questionnaires related to altered states of 
consciousness and psychometrics.  
STATISTICAL ANALYSIS 
Functional connectivity magnetic resonance imaging (fcMRI) 
fcMRI will be performed as described in our previous publications.5, 8, 15 During fcMRI, participants will 
either be at rest or receiving a pain stimulus via a pressure cuff applied to the left lower leg evoking 
tonic pressure with pain of 40 on a 100 point numerical rating scale (0=no pain; 100=worst pain 
imaginable). This sustained pain is relevant to deep musculoskeletal clinical pain and can be applied for 
long durations (i.e. minutes) without tissue damage.16  Data will be corrected for physiological effects 
such as cardio-respiratory fluctuations that are known to influence fcMRI estimation.17, 18 Resting 
connectivity network analysis will be performed using the FSL (FMRIB’s Software Library, 
www.fmrib.ox.ac.uk/fsl) software package. All data will be corrected for motion artifacts, smoothed 
(FWHM 5 mm), and high-pass temporal filtered (f = 0.006Hz). ICA Approach. The within- and between-
subject resting fMRI data analysis will be performed using ICA through Multivariate Exploratory Linear 
Optimized Decomposition into Independent Components (MELODIC, an FSL tool) and a previously 
validated dual regression approach.19 We will apply probabilistic ICA to identify global, independent 
patterns of functional connectivity existent in the entire subject population. The number of independent 
components will be limited to 25 to reduce subcomponents. Networks of interest will be selected and 
the spatial independent component maps identified from the population data will be used as a spatial 
regressor in a general linear model of the subject’s resting fcMRI data. This will allow us to estimate 
subject-specific spatial maps for each component. These maps will then be passed up to group analysis. 
Seed-voxel Approach: We will evaluate functional connectivity using seed-voxels from brain regions 
previously shown to be involved in consciousness or pain. The average time course in these regions will 
be used as regressors in a whole brain general linear model to find other brain regions also correlated 
with the seed region. A whole brain general linear model will be performed using the seed time course, 
averaged over a 3 mm radius sphere, as the regressor of interest. The resulting single subject maps will 
be normalized to standard space and passed up to group analyses. fcMRI group analyses: Nitrous oxide 
effects on brain connectivity will be estimated using a factorial model with treatment (nitrous versus 
placebo) and time (pre versus post) as factors. All statistical maps will be corrected for multiple 
comparisons on the cluster level p < 0.05, derived from an uncorrected p < 0.001 on the voxel level. 
Network Analysis 
In terms of network properties to be analyzed, the degree is the number of connections a node (in this 
case, a given brain region) has, while the modularity is a measure of the extent to which the connected 
regions form individual functional modules. We will first investigate the characteristic modules 


Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
corresponding to different states, then quantify the modularity of the networks. High-degree hubs 
facilitate “shortcuts” of network information transmission (much like a hub airport facilitates travel 
shortcuts) and are identified with the measures of centrality in a network. The centrality of a node 
measures its relative ability to integrate information globally within a brain network. We will assess 
several centrality measures (degree centrality, closeness centrality, betweenness centrality and 
Eigenvector centrality) to test the robustness of the hub structure. The average path length, which 
measures the mean number of steps along the shortest paths for all possible pairs of network nodes, 
will be used for quantifying the global efficiency of brain networks (since efficiency varies inversely with 
path length). Network measures will be compared across states (waking, analgesia, anesthesia) using a 
repeated measures two-way analysis of variance (ANOVA) and a post hoc Tukey multi-comparison test 
(behavioral state as the within-subjects factor, anesthetic agent as the between-subjects factor). A p 
value <0.05 will be considered significant. 
EXPECTED OUTCOMES 
Based on preliminary data, we predict that administration of low-dose nitrous oxide will decrease 
network modularity and increase network efficiency, disrupting normal pain processing. Specifically, we 
expect to see altered relationships of key pain structures (insula, thalamus, S1 and S2) and their 
connections with the DMN. We expect that, as cortical activation and disorganization increase with 
higher doses, nitrous oxide will increase network modularity and decrease network efficiency, disrupting 
conscious processing of the environment in general. Our prior study of propofol44 and preliminary data 
with 60% nitrous oxide showing decreased efficiency and increased modularity suggest the exciting 
possibility that GABAergic and non-GABAergic anesthetics share common network-level mechanisms to 
induce unconsciousness. 
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Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
APPENDIX A 
ETHNIC / RACIAL TARGETED ENROLLMENT TABLE 
 
Study Title: Network-Level Mechanisms of Nitrous Oxide in the Human Brain 
 
Total Planned Enrollment: 22 
 
TARGETED/PLANNED ENROLLMENT: Number of Subjects 
Ethnic Category 
Females 
Males 
Total 
Hispanic or Latino 
1 
1 
2 
Not Hispanic or Latino 
10 
10 
20 
Ethnic Category: Total of All Subjects * 
11 
11 
22 
Racial Categories 
 
American Indian/Alaska Native 
0 
0 
0 
Asian 
2 
2 
4 
Native Hawaiian or Other Pacific Islander  
0 
0 
0 
Black or African American  
2 
2 
4 
White 
7 
7 
14 
Racial Categories: Total of All Subjects * 
11 
11 
22 
* The “Ethnic Category: Total of All Subjects” must be equal to the “Racial Categories: Total of All Subjects.”