Clinical trial NCT01794689
Conditions: Sleep Deprivation Pain
Sponsor: Johns Hopkins University
Start date: 2013-05-01; Completion date: 2019-03-12
Enrollment: 100 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
Twenty percent of Americans suffer from chronic pain. Sleep disturbance is similarly prevalent and among the most common and disabling neurobehavioral problems associated with chronic pain. This research is designed to evaluate the effects of disrupted sleep patterns on mood, inflammation, the perception of pain, and pain relief. This study will help researchers understand the relationship between sleep and pain, and how sleep disturbance might influence chronic pain conditions.

Detailed description:
This research is being conducted in order to evaluate the effects of disrupted sleep patterns on mood, inflammation, the perception of pain, and pain relief. This study will help researchers understand the relationship between sleep and pain, and how sleep disturbance might influence chronic pain conditions. Healthy participants will undergo baseline sleep and sleep disruption conditions. Following undisturbed sleep and sleep disruption conditions, sensitivity to pain and analgesic response (via morphine or placebo administration) will be assessed using a heat-capsaicin pain model.

This study will be conducted in 2 major parts-3 screening visits (2 outpatient and 1 inpatient) and 2 experimental inpatient visits. Part 1 of the study will involve a 1-week screening period. This will involve two separate screening visits lasting about 2 hours each. At Screening Visit 1, participants will complete questionnaires, an interview, and undergo toxicology screening. At Screening Visit 2, participants will complete questionnaires, undergo a physical exam, and be familiarized with pain testing procedures. At Screening Visit 3, participants will undergo an inpatient sleep study.

Part 2 will involve two different inpatient admissions. The two admissions will be separated by at least two weeks. During each of the admissions, participants' sleep will be studied at night. The first admission will begin immediately following the overnight sleep study in Screening Visit 3. One of the admissions will be for one night and the other admission will be for three nights. For the one night admission, participants will sleep undisturbed for an 8-hour period. For the three night admission, participants will undergo sleep disruption for two nights in a row. On the third night, participants will be allowed to sleep undisturbed for 8 hours for recovery.

During both inpatient admissions, pain testing procedures will be completed that will last approximately 5 hours during the day. During testing, small amounts of blood will be drawn for analysis. Participants will be randomly assigned to two groups. Group A will be given a standard dose of morphine during pain testing. Group B will be given a placebo during pain testing.

Arms: arm 1: Participants randomized to receive Morphine and the Uninterrupted Sleep (US) condition first. After a polysomnography (PSG) screening night, participants were randomized to receive two consecutive nights of uninterrupted sleep (US). With a minimum of a two week washout period, participants then completed the opposing sleep condition of two nights of forced awakenings (FA). They will receive the Morphine injection (0.08mg/kg) via IV bolus over 30 seconds during each experimental quantitative sensory testing session that occurs after the US and FA sleep conditions are completed. arm 2: Participants randomized to receive the saline placebo and the Uninterrupted Sleep (US) condition first. After a polysomnography (PSG) screening night, participants were randomized to receive two consecutive nights of uninterrupted sleep (US). With a minimum of a two week washout period, participants then completed the opposing sleep condition of two nights of forced awakenings (FA). They will receive the injection via IV bolus over 30 seconds during each experimental quantitative sensory testing session that occurs after the US and FA sleep conditions are completed. arm 3: Participants randomized to receive Morphine and the Forced Awakenings (FA) condition first. After a polysomnography (PSG) screening night, participants were randomized to receive two consecutive nights of forced awakenings (FA). With a minimum of a two week washout period, participants then completed the opposing sleep condition of two nights of uninterrupted sleep (US). They will receive the Morphine injection (0.08mg/kg) via IV bolus over 30 seconds during each experimental quantitative sensory testing session that occurs after the FA and US sleep conditions are completed. arm 4: Participants randomized to receive the saline placebo and the Forced Awakenings (FA) condition first. After a polysomnography (PSG) screening night, participants were randomized to receive two consecutive nights of forced awakenings (FA). With a minimum of a two week washout period, participants then completed the opposing sleep condition of two nights of uninterrupted sleep (US). They will receive the injection via IV bolus over 30 seconds during each experimental quantitative sensory testing session that occurs after the FA and US sleep conditions are completed.
Interventions: intervention 1: Morphine intervention 2: Saline Placebo intervention 3: Forced Awakenings intervention 4: Uninterrupted Sleep
Intervention descriptions: intervention 1: 0.08mg/kg will be administered to participants randomly assigned to receive the drug via IV bolus during each quantitative sensory testing session (after one night of uninterrupted sleep and after 2 nights of forced awakenings). intervention 2: Saline Placebo will administered to participants randomly assigned to receive the placebo via IV bolus during each quantitative sensory testing session (after one night of uninterrupted sleep and after 2 nights of forced awakenings). intervention 3: Participants will be awakened each hour during an 8 hour sleep opportunity period. One of the awakenings is for 60 minutes and randomly determined. The other 7 awakenings are for 20 minutes each, and are randomly scheduled to occur in either the first second or third tertile of each hour. The maximum total sleep time a participant will receive is 280 minutes. intervention 4: Participants will receive an 8 hour period of undisturbed sleep

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
Mechanisms of Sleep Disruption Hyperalgesia 
NCT 01794689 
August 30, 2016 
 


Date: 08/30/16Principal Investigator: Michael T. Smith, Ph.D. 
Application Number: NA_00071465 
JHMIRB eFormA  01 
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Johns Hopkins Medicine - eForm A 
 
 
 
 Use the section headings to write the eForm A, inserting the appropriate material in each. If a 
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 When submitting eForm A (new or revised), enter the date submitted to the field at the top of 
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1. 
Abstract 
Twenty percent of Americans suffer from chronic pain. Sleep disturbance is similarly prevalent and 
among the most common and disabling neurobehavioral problems associated with chronic pain. 
Hyperalgesia is a pathophysiologic feature of chronic pain syndromes. In this study, we propose to 
initiate a systematic investigation of the mechanisms by which sleep disruption incites hyperalgesia 
(HA). Addressing this knowledge gap, by identifying the mechanisms of sleep disturbance-induced 
hyperalgesia (SD_HA) has critical implications for the etiology, prevention and treatment of chronic 
pain. Preliminary data from our investigative teams implicate two untested and possibly interrelated 
candidate mechanisms: 1) inflammation and 2) opioidergic antinociceptive system impairment. We 
have found that experimental sleep restriction activates cellular and genomic markers of 
inflammation; based on non-human animal data, we hypothesize inflammation will heighten 
hyperalgesia. We have also shown that experimental sleep disruption impairs descending pain 
inhibition, causes spontaneous pain, and diminishes opioid analgesia. Despite these convergent data, 
whether and to what degree SD_HA is mediated by inflammation is unknown. We have assembled 
an interdisciplinary team from Johns Hopkins and UCLA to tackle the problem of SD_HA. We 
propose a translational experiment in healthy human subjects to determine the role of inflammation 
in SD_HA and study the effects of sleep disruption and inflammation on opioid analgesia.  Using a 
mixed-model, randomized controlled experiment, all participants will undergo baseline sleep and 
experimental sleep disruption conditions. We will employ a novel sleep fragmentation manipulation 
developed by our group, as an analogue for the type of sleep loss most commonly associated with 
pain and insomnia—multiple nocturnal awakenings. Following undisturbed sleep and sleep 
disruption conditions, we will examine next-day hyperalgesia and analgesic response to either: (a) 
morphine or (b) placebo. Hyperalgesia and analgesia will be assessed using a heat-capsaicin pain 
model, which provides an index of spinally mediated hyperalgesia [secondary hyperalgesia (2o HA)], 
yet applied to the study of SD_HA. Candidate inflammatory markers will be measured at the: 1) 
genomic  2) cellular and 3) systemic levels of analysis, and will be evaluated as mediators of the 
effects of sleep disruption on hyperalgesia and opioid analgesia.  
 
2. 
Objectives (include all primary and secondary objectives) 
 
AIM 1: To examine the effects of experimental sleep disruption on spinal sensitization by 
evaluating laboratory pain responses in the heat-capsaicin pain model. 
 
Hypothesis 1.a. Relative to uninterrupted sleep, experimental sleep disruption will enhance primary  
hyperalgesia (1o HA) and 2o HA (an index of central neuronal sensitization).  
 
AIM 2:  To examine the effects of experimental sleep disruption on opioid analgesia. 


Date: 08/30/16Principal Investigator: Michael T. Smith, Ph.D. 
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Hypothesis 2.a. Relative to undisturbed sleep, sleep disruption will diminish the analgesic effect of 
morphine on 1o HA, 2o HA (heat-capsaicin pain model), and cold pressor pain tolerance (CPT). 
 
AIM 3:  To determine the effects of sleep disruption on cellular and genomic markers of 
inflammation and characterize the role of inflammatory activity on laboratory pain responses 
and opioid analgesia. 
 
Hypothesis 3.a. Relative to undisturbed sleep, experimental sleep disruption will increase markers 
of inflammation across genomic, cellular, and systemic levels of analysis.  
Hypothesis 3.b. Markers of inflammation will mediate the effects of experimental sleep disruption 
on 1o HA and 2o HA, and diminished morphine analgesia on 1o HA,  2o HA, and CPT. 
 
3. 
Background (briefly describe pre-clinical and clinical data, current experience with procedures, 
drug or device, and any other relevant information to justify the research) 
 
Twenty to thirty-five percent of the world population suffers from chronic pain syndromes(1-3) 
at an estimated cost of $100 billion per annum in the US alone(4). In addition, persistent pain 
profoundly impairs quality of life(5, 6) and increases risk for medical(7) and psychiatric morbidity(8). 
Chronic pain is conceptualized as a disease of the nervous system, involving varying degrees of 
peripheral input and dysregulation of central nociceptive modulatory systems(9, 10). Central 
neuroplastic alterations amplify and maintain nociception leading to generalized hyperalgesia and 
clinical pain that can persist disproportionately to or in the absence of peripheral damage. 
Hyperalgesia, i.e. heightened responsivity to noxious stimuli, characterizes chronic pain syndromes 
and is critical to the pathogenesis and treatment of chronic pain(11). The complex interactions 
between behavioral, environmental and neurophysiologic processes that promote and maintain 
hyperalgesia, however, are just beginning to be explored and remain poorly understood. 
Insufficient sleep is one promising, modifiable behavioral factor with major relevance to the 
study of hyperalgesia and chronic pain. Sleep disruption due to chronic insomnia is the most 
common clinical cause of sleep loss with a prevalence between 10-15% (12-15), but a substantial 
number of individuals obtain insufficient sleep from self-imposed curtailment, lifestyle, and work-
related factors(16). The CDC reports that almost one third of US adults experience insufficient sleep 
for the majority of days in the preceding month(17).  
Sleep disruption is one of the most disabling comorbidities reported by 50-88% of chronic pain 
patients(18, 19). Several controlled experiments demonstrate that sleep deprivation causes 
hyperalgesia(20-22) and longitudinal data indicate that sleep disruption and clinical pain reciprocally 
interact in a vicious cycle(23-27). Exacerbations in pain predict decrements in sleep and poor sleep in 
turn predicts subsequent pain(28). Sleep disturbance increases the risk for developing chronic pain 
after acute traumatic injury(29, 30) and enhances the odds of developing widespread pain(31, 32). 
Conversely, restorative sleep increases the likelihood of remission from chronic pain(33). These 
findings, which are independent of psychological factors, such as negative mood, suggest that sleep 
disruption may play a critical role in the poorly understood transition from acute to chronic pain. 
Since sleep disorders and insufficient sleep are highly prevalent and treatable, the potential 
impact of systematic research in this area is substantial and may enhance our understanding 
of chronic pain pathophysiology, as well as lead to novel pain prevention and management 
approaches.   
The mechanisms of sleep disruption-induced hyperalgesia (SD_HA) are unknown, but our 
previous work implicates functional alterations of supraspinal, descending pain modulatory 
sytems(34), in which opioid peptides play a critical mediating role(35-41). Compromised pain 


Date: 08/30/16Principal Investigator: Michael T. Smith, Ph.D. 
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inhibitory capacity, psychophysiologically measured in humans using diffuse noxious inhibitory 
control (DNIC) testing paradigms, has been demonstrated in many idiopathic clinical pain 
conditions(42-48), such as fibromyalgia. One way to probe the mechanisms underlying sleep 
dependent impairment to these systems, with great potential for significance, is to determine 
whether sleep disturbance reduces the effectiveness of exogenously administered opioids.  
Supported by preclinical studies95;96 and our pilot data (see below), we propose to determine 
whether sleep disruption interferes with opioid analgesia in humans (AIM2). Such a finding 
would have broad implications for clinical practice and conceivably influence the care of millions 
of patients. Opioid receptor agonists are the standard treatment for moderate to severe acute pain. 
Approximately16% of all emergency room visits result in the prescription of opioid analgesics(49) 
and long-term opioid therapy is increasingly used to treat non cancer-related chronic pain(50). 
Establishing the impact of disrupted sleep, a highly treatable condition, on opioid efficacy, 
therefore, could lead to more efficient, effective, and safer use of opioids to treat pain and 
potentially reduce risk of overuse and dependency. Inadequate pain management continues to be a 
major problem in the US(51), despite data demonstrating that poorly controlled pain increases the 
length of hospital stays and contributes to poor outcomes(52, 53). Positive findings from the 
proposed study, therefore, could potentially change clinical practice guidelines to include 
proactively identifying and treating sleep disturbance via behavioral and/or pharmacologic 
interventions before or in conjunction with opioid therapy to improve pain management outcomes.  
Our focus on inflammation as another key mediator of SD_HA also has broad implications 
for chronic pain prevention and treatment. Sleep deprivation elevates pro-inflammatory substances, 
which are known to sensitize nociceptors and induce enhanced responsivity to noxious stimulation 
in animals. It is not known whether increased inflammation induced by sleep deprivation mediates 
hyperalgesia in humans. By investigating multiple levels of the inflammatory cascade, including 
intracellular cytokine production, genomic and molecular signaling pathways, our approach 
provides a finer grained understanding of the mechansims by which sleep disruption alters 
inflammatory processes. Beyond connecting alterations in inflammatory pathways to hyperalgesia 
in humans, this knowledge also has broader health implications for many chronic conditions such as 
cardiovascular disease(54, 55) and autoimmune disorders(56) in which rates of sleep disorders are 
high and chronic inflammation plays a substantial role in morbidity. 
Lastly, we will evaluate possible interrelationships between opioid efficacy and 
inflammation, determining whether inflammation mediates (statistically) sleep disruption’s 
attenuation of opioid analgesia. This hypothesis derives from recent animal studies(57, 58) and 
translation would have major implications for pain medicine, including the development of novel 
analgesics. If SD-induced inflammation does interfere with opioid analgesia, this provides a 
scientific basis for clinical reports that combining anti-inflammatory agents with opioid analgesia 
reduces post operative opioid consumption and enhances surgical outcomes(59). 
Summary. The proposed project addresses two major public health epidemics—insufficient 
sleep and chronic pain. Each aim has the potential to change the clinical practice of pain prevention 
and management. The scientific knowledge gleaned by our detailed study of the mechanisms of 
SD_HA—including genomic and cellular dimensions of the inflammatory cascade, opioidergic 
function, and their interaction—has broad implications for some of the most costly and prevalent 
chronic diseases, including opioid addiction, cardiovascular disease, and rheumatologic disorders. 
 
 
4. 
Study Procedures 
a. Study Design  
 
RESEARCH DESIGN AND METHODS  
 
 
 
 
 
 
     


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Design:  As depicted in Figure 1, we have adopted a mixed model, randomized controlled, double-
blinded, experimental design that combines the feasibility of a parallel, between-groups approach 
[Drug condition (morphine vs. placebo saline)] with the statistical power advantages and cost 
effectiveness of a within subjects approach. Sleep is the within subjects condition [Uninterrupted 
Sleep (US) vs. Forced Awakenings (FA)]. The US condition is 2, 8-hour nights of undisturbed sleep 
(US1 and US2), whereas the FA condition involves two consecutive nights (FA1 & FA2) of 8 
forced awakenings plus a recovery night (not shown in diagram). Eligibility will be determined by 
three screening visits, including an adaptation polysomnography (PSG) assessment [Night1 (v3)]. 
Subjects are randomized (Morphine or Placebo) and will then complete two separate admissions (v4 
& v5) at the JHBMC CRU to undergo the US and FA conditions and next day assessments. An 
optional testing session (see Optional Sleep Pain, Emotions, and Reward Study Procedures, below) 
will occur in the daytime after US1 and FA1. The order of sleep condition admissions will be 
counterbalanced and there will be a ≥two week washout period between admissions. The daytime 
pain testing protocol (Cap/QST & drug) will be performed twice, the day after US2 and the day 
after two nights of sleep disruption (FA2). The protocol includes blood sampling to quantify 
inflammatory markers, heat-capsaicin sensitization, quantitative sensory testing (QST), and drug 
administration. Five QST sessions will be conducted to assess HA and morphine analgesia (1 pre-
drug and 4 post-drug; details provided below and in Fig. 3).  Morphine/placebo will be administered 
through subcutaneous injection after the completion of QST1. There will be a 40 minute rest period 
following morphine/placebo administration to allow for peak analgesic effect to take place. 
Subsequently QST 2-5 (i.e., rekindling) will be conducted at 40 minute intervals. 
 
Optional Sleep, Pain, Emotions, and Reward Study Procedures 
 
Participants will have the option to participate in the Sleep, Pain, Emotions, and Reward Study 
(SPEAR). The purpose of this optional study is to evaluate the influence of sleep continuity disruption on 
emotion reactivity and regulation, as well as motivation and reward processing. This study is embedded 
within the Mechanisms of Sleep Disruption Hyperalgesia Study, such that participants will undergo the 
same screening procedures.  Eligibility criteria for the SPEAR study will be similar to the Mechanisms of 
Sleep Disruption Hyperalgesia Study. Subjects participating in SPEAR will complete an additional consent 
form for these procedures. Consent will be obtained in advance, along with the Parent study consent 
procedures. The procedures for participants in the SPEAR Study will differ from those associated with the 
Mechanisms of Sleep Disruption Hyperalgesia Study in the following ways: 
 
 
1) On the day following Night 1 of the US condition (US1), and on the day following Night 1 of 
the FA condition (FA1), participants in the SPEAR study will complete a series of emotion, 
motivation, reward-related, and psychomotor vigilance tasks, as well as additional pain testing. 
Some of these tests will occur simultaneously. It is expected that the experimental procedures 
associated with SPEAR will last approximately 2-3 hours (see below for detailed procedures 
related to the optional SPEAR study). 
2) Participants will have the option of participating in one magnetic resonance imaging (MRI) scan 
along with two positron emission tomography (PET) scans. We are including PET imaging in 
this protocol in order to evaluate the extent to which dopamine receptor 2/3 binding correlates 
with the emotion and reward-related behavioral data we are already collecting as part of the 
SPEAR substudy, which is a natural extension of our ongoing work. 
 
A study design of the entire study (Mechanisms of Sleep Disruption Hyperalgesia + SPEAR) is provided in 
Figure 1 below: 


Date: 08/30/16Principal Investigator: Michael T. Smith, Ph.D. 
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Figure 1. Study Timeline When SPEAR Study is Embedded in Mechanisms of Sleep Disruption 
Hyperalgesia Study 
 
 
ASSESSMENT PROCEDURES  
 
Recruitment: We will recruit subjects via community flyers, print, and electronic media. 
 
Screening (phone and  in person  Visit 1):  A phone screen eliciting information relevant to the 
inclusion/exclusion criteria will be conducted, adapting our current screens for the proposed study.  
After passing the initial phone screen, subjects will complete a 2 hour visit at the JHU Behavioral 
Sleep Medicine Lab, which will include: informed consent, a urine toxicology screen, and 
anthropometry. We will administer the following standardized questionnaires: The Pittsburgh Sleep 
Quality Index [(PSQI)(60)], the Epworth Sleepiness Scale (ESS)(61), the Brief Symptom Inventory 
(BSI)(62), the Brief Pain Inventory (short form) (BPI)(63), and a Health History Form (HHF)(64). 
The ESS and PSQI, have well established cut-off criteria demarcating excessive daytime 
sleepiness(65) and poor sleep(60, 66). The BSI global scales are well normed indicators of general 
psychological distress(67). The HHF obtains detailed information related to medical disorders, pain 
history, medications and health behaviors (caffeine, exercise, etc.) that is supplemented by 
interview to establish eligibility. Subjects will complete the Structured Clinical Interview for DSM-
IV Psychiatric Disorders (SCID-DSM-IV Patient Questionnaire)(68) to identify likely Axis I 
disorders, including substance abuse. Follow up administration of selected, full SCID-IV Axis I(68) 
modules will determine psychiatric eligibility. The Structured Interview for Sleep Disorders(69) 
will be used to exclude sleep disorders. The Time-line Follow-Back (TLFB) procedure(70) will 
obtain opioid use history. Subjects will be trained on the Sleep and Pain Diaries and Wrist 
Actigraphy procedures. 
 
Screening Visit 2: Subjects will keep standard daily sleep(71-73) and pain diaries(74) for 1 week. 
Morning entries gather sleep continuity parameters. Evening entries query for daytime pain(63), 
fatigue, naps, medications, menstruation, and exercise. Subjects will continuously wear an 
ActiSleep Plus Monitor (or equivalent) wrist accelerometer, which provides objective estimates of 
circadian rhythm,(75) and sleep continuity(76, 77). Sleep diary and actigraphy data will be 
reviewed to determine that the sleep, pain, and other eligibility criteria are met. In addition, 
participants will have the option of providing a saliva sample to be used for future genetic analyses.  
Should participants accept this option, 1.0 mL of whole unstimulated saliva will be collected by 
passive drool using a saliva collection aid (e.g., Oragene DNA Self-Collection Kit), stored 
according to standard specifications.  In addition, subjects will have a history and physical exam 


Date: 08/30/16Principal Investigator: Michael T. Smith, Ph.D. 
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and must test negative for pregnancy via urine pregnancy test (females) and recreational drugs and 
opioids. A complete blood count test, hepatic and kidney function panel will be performed. Drs. 
Tompkins, Strain, or Umbricht will make final medical eligibility determinations. 
 
Subjects will be briefly familiarized with the pain testing procedures and capsaicin cream to further 
help determine eligibility. A thermode will be secured to the forearm with a Velcro strap and will be 
programmed to emit heat at a constant temperature (e.g., 45°C) for 5 minutes. The thermode will 
then be removed and .1% capsaicin cream will be applied to the area on which the thermode rested, 
and will be covered with a raised adhesive patch. Subsequently, the patch and capsaicin will be 
removed and the skin will be cleaned with an alcohol swab. Participants who find the pain from the 
capsaicin treatment to be intolerable, or who lack an adequate pain response, will be excluded from 
further participation in the study. These criteria are in place to avoid causing undue pain (e.g., those 
whose pain ratings are already quite high would presumably experience even more pain during 
subsequent quantitative sensory testing over the affected area) and to rule out those whose 
“baseline” pain scores are too low to assess opioid analgesia. 
 
Screening Visit 3 [inpatient Clinical Research Unit (CRU)]: Twenty-four hours prior to, and during 
admission for Visit 3 (v3) and all CRU visits, subjects will refrain from taking analgesics, 
hypnotics, and other centrally acting (e.g., caffeine) or anti-inflammatory agents. Subjects will 
undergo a standard PSG, as described below, to rule out occult sleep disorders and provide an 
adaptation night. Subjects meeting ICSD Criteria(78) for a sleep disorder (e.g. sleep apnea) will be 
ruled out and referred for clinical care. 
 
Randomization Plan. Subjects passing v3 will remain on the unit and be randomized to receive 
either morphine or placebo during both of their US and FA admissions (v4 & v5). The Research 
Pharmacy will generate the randomization list to maintain double-blinding. We will use a dynamic 
probabilities algorithm with minimization(79, 80) to balance groups on sex, obesity (BMI), and age, 
since these factors influence pain and inflammation. We will also balance on Chinese ethnicity, due 
to its association with morphine metabolism(81). To control for sequence effects, we will 
counterbalance US and FA conditions by randomizing subjects to 1 of 4 sequences: 1) Placebo/US 
first; 2) Placebo/FA first; 3) Morphine/US first and 4) Morphine/FA first. Subjects randomized to 
FA first, will remain inpatient for 3 consecutive nights [(v4) FA1, FA2, Recovery]. After two weeks 
washout, they will complete their US admission(v5). Subjects randomized to the US sequences first 
will complete the US admission [(v4) US1, US2] followed two weeks later by their FA admission 
[(v5) FA1, FA2, & Recovery]. 
 
Experimental Assessments.   
 
General Inpatient CRU Procedures.To maintain experimental control, subjects will not leave the 
unit during each admission (v3-5) and will not be permitted to sleep outside their prescribed 
nocturnal schedule. Staff will continuously monitor, maintain, and regularly document wakefulness. 
Subjects will wear actigraphs at all times during v3-5 to provide an additional, objective procedural 
integrity check. Standardized meals and snacks will be provided by the CRU. Breakfast will be 
served at approximately 7:30 AM. Subjects will continue to complete sleep and pain diaries during 
v3-5 to provide indices of spontaneous pain and fatigue. 
 
Questionnaires. Each inpatient evening, subjects will complete the Stanford Sleepiness Scale, to 
measure subjective changes in sleepiness(82) and the Positive Affect Negative Affect Schedule – 
Extended (83) to measure general affective dispositions and discreet emotions. At baseline only, 


Date: 08/30/16Principal Investigator: Michael T. Smith, Ph.D. 
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participants will complete a battery of questionnaires including the Temporal Experience of 
Pleasure Scale (84) to measure anticipatory and consummatory anhedonia, the Behavioral 
Inhibition/Behavioral Activation Scale (85) to measure approach-oriented motivation, the Trait 
Meta-Mood Scale (86) to assess attention to one’s own mood, and the ability to regulate positive 
and negative moods, and the Monetary Choice Questionnaire to assess delay discounting (87). 
Research participants will also complete brief computerized cognitive (assessing attention, 
concentration, and memory) and motivation tasks following the experimental sleep manipulations. 
 
Optional Sleep, Pain, Emotions, and Reward (SPEAR) Study Procedures 
Participants will complete several tasks assessing perceptual (e.g., reaction time), emotional, 
and psychophysiological (e.g., facial electromyography; galvanic skin response) responses to the 
presentation of emotionally evocative images taken from the standardized and widely used bank of 
the International Affective Picture System (88). In addition, participants will complete tasks 
assessing their responses to the presentation of real and hypothetical monetary or food rewards, 
food reward questionnaires, and a task assessing psychomotor vigilance. 
Participants will also undergo quantitative sensory testing (QST) procedures conducted in 
the parent project to assess responses to heat, pressure, and cold stimuli (e.g., pain threshold, 
tolerance, intensity). The heat and pressure pain threshold and cold pressor procedures are the same 
as those conducted in the Mechanisms of Sleep Disruption Hyperalgesia study, and are described in 
greater detail below. Additionally, heat stimuli will be presented simultaneously with cognitive, 
emotion and reward-related tasks. 
Additionally, participants (N = 15) will have the option to participate in a PET imaging 
substudy, which will involve an MRI scan and two PET scans (one after each sleep condition). 
MRI  
A limited MRI of the brain (without contrast) will be obtained at JHOC for future 
anatomical co-registration with the brain PET scan. The MRI exam will take approximately 20-30 
minutes. Incidental findings on MRI scans that are pathognomonic for an active disease or 
pathological process which requires medical intervention will be exclusionary. Other incidental 
findings, such as unidentified bright objects (UBOs), most venous vascular anomalies, benign cysts, 
heterotopic rests of neurons, and anatomical variants that are known to be prevalent in the general 
population of healthy people, will not be exclusionary. Unexpected findings of concern will be 
reviewed by a qualified neuroradiologist in consultation with the investigators to determine the 
subject’s eligibility for participation in the study in light of these findings. If the MRI shows any 
clinically significant abnormalities, the participant will be notified. 
PET Scans: 
In the morning following the first night of each sleep condition (US and FA), subjects will 
be transported from the CRU at JHBMC to the Johns Hopkins PET Center for the scheduled PET 
scan.  
 
Prior to the first PET scan, subjects will have a facemask constructed to facilitate 
maintaining the same position of the head for the PET scans. The facemask will be made prior to 
the first PET scan and will not require an additional visit. The facemask will take approximately 10 
minutes to construct and it will be used during the scanning. 
 
Before each PET scan a catheter will be inserted into a vein of one arm for the injection of 
the radiotracer.  
After the catheter is placed the participant will be placed in the PET scanner. A 6 to 10 
minute transmission (attenuation) scan will be performed before or after the PET scan.  Participants 
will undergo a PET scan lasting approximately 90 minutes after the intravenous bolus injection of 


Date: 08/30/16Principal Investigator: Michael T. Smith, Ph.D. 
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approximately 20 mCi [11C]raclopride. Dynamic PET data will be acquired during scanning. 
Continuous single lead ECG will be measured throughout the PET imaging. If during this period 
abnormal single ECGs are detected,  12-lead ECG will be recorded. After the completion of the 
PET scan, subjects will remain at Johns Hopkins under medical supervision. Subjects will be 
discharged if deemed safe to be discharged by the clinical investigator. 
 
Nocturnal Polysomnography (PSG). PSGs will be conducted on all experimental nights (1-4) in 
private rooms. Lights out will occur at the subject’s average bedtime per diary and actigraphy. 
Registered PSG technicians (RPSGT) will execute the PSG procedures according to standard 
technical guidelines(89). The recording montage uses the AASM(89) recommended placement for 
EEG, EOG, EMG and ECG. We will acquire electrophysiologic signals using an Embla N7000 
polygraph. Respiratory function and effort will be measured via oronasal thermistor, nasal air 
pressure transducer, pulse oximetry, and abdominal and thoracic plethysmography belts. After 
Night1, we will abbreviate the montage for comfort, removing anterior tibialis EMGs and all 
respiratory sensors. Records will be scored according to AASM guidelines, by an RPSGT and 
reviewed by a physician, board certified in sleep medicine. Both will be blind to study hypotheses. 
  
Experimental Sleep Conditions. 
 
Forced Awakenings Condition.The FA condition will be conducted on two consecutive nights 
as previously described(34). An 8-hour sleep opportunity period starting from lights out is divided 
into eight, one-hour intervals. One of the intervals is randomly determined to be a 60 minute forced 
awakening, during which no sleep is permitted. Each of the remaining seven, 60 minute intervals 
are subdivided into tertiles (20 min. blocks). A forced, 20 min awakening is randomly scheduled to 
occur in either the 1st, 2nd, or 3rd tertile of each hour. During forced awakenings, staff keep subjects 
awake, either by voice or gentle shaking. Subjects sit up in bed, but lights remain off. Techs, 
monitoring PSG signals, inform the attendant if the subject starts to fall asleep. PSG monitoring is 
maintained for the entire sleep period. The maximum total sleep time possible, if subjects sleep 
100% of the time when not forced awake (7, 20-min & 1, 60-min), is 280 minutes. 
 
Uninterrupted Sleep Condition. An 8 hour period of undisturbed sleep is permitted. 
 
Optional Sleep, Pain, Emotions, and Reward Study Procedures 
 
A second night of undisturbed sleep will be added for participants who elect to participate in 
the optional study. 
 
Blood Draws, Pain Testing & Drug Protocol. Figure 3 depicts the timing of blood draws, heat 
capsaicin procedures, quantitative sensory testing, and drug administration. This protocol will be 
implemented in the mornings after the US night and the second FA night (FA2). Drs. Smith and 
Campbell have experience conducting all of the pain testing procedures(90-93). Drs. Strain, 
Tompkins, and Umbricht have extensive expertise studying the effects of opioid agonists(94, 95). 
All pain testers complete a rigorous training process that includes inter-rater reliability testing 
(kappa>.80). Participants are permitted to terminate any of the procedures at any time. The 
following procedures will be conducted in an identical fashion on both visits. All assessments will 
be conducted by BSMP staff, who will be blind to subject conditions.  


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Blood Draws & Handling Procedures. Subjects will remain in a semi recumbent position during the 
procedure. Approximately 30 minutes after Lights On (i.e., wake), the nurse will insert a single port 
lumen peripheral catheter in an upper extremity vein, contralateral to the arm used for capsaicin 
testing. A continuous saline infusion at 250cc/h will be maintained.  The line will be flushed with 
saline before and after all blood draws, and will remain installed until the final QST procedure is 
complete. As shown in Figure 7, seven blood draws will be obtained (3 pre-injection and 4 post-
injection). Blood draws 1 and 2 will be obtained approximately 60 minutes apart, prior to the 35 
minute heat capsaicin procedure, to provide a resting baseline. After the initial capsaicin procedure, 
all blood will be drawn prior to each rekindling. Blood will be immediately transported to the 
Rheumatology Disease Research Core for immediate preparation by the RDRC tech. 
 
Heat-Capsaicin and Rekindling Procedures. We will utilize procedures similar to established 
protocols(96, 97). Heat will be delivered using a computer driven peltier heating element (Medoc 
TSA II). The procedure involves a 35 minute sensitization period. A 10.24 cm2 treatment site, the 
size and shape of the thermode will be randomly assigned and marked on either a lower or upper, 
non overlapping surface of the ventral forearm opposite the arm from which blood is drawn. Lower 
and upper treatment sites will be counterbalanced across admissions. The thermode is secured on 
the skin with velcro straps and heated at 45°C for 5 minutes. Pain ratings will be collected once 
every minute on a 0-100 VAS (“no pain”, “most extreme pain imaginable”). An open square, raised 
adhesive frame patch (internal dimensions same as thermode) is immediately applied to the borders 
of the treatment site and Capsaicin cream (0.1% Capsaicin)(90) is evenly spread onto the skin and 
permitted to absorb for 30 minutes. This induces moderate (Mean = 48.25+ 27.93), but well 
tolerated pain. The raised frame prevents spillage or leakage of capsaicin outside the treatment site. 
Capsaicin is removed, blood draw 3 is obtained, and QST1 is performed as described below. 
Following morphine/placebo injection and the subsequent 40 minute rest, the treated skin will be re-
kindled a total of four times at 40 min intervals by heating the treatment site with the thermode at 
40°C for 5 min. Following each rekindling episode, a series of QST procedures to quantify 1o and 2o 
Hyperalgesia will be conducted. Studies have demonstrated excellent test-retest stability in 
hyperalgesia measurements over 4 rekindlings(96, 97, 97). The timing associated with these 
procedures may vary as a function of experimental demands. 
 
Quantitative Sensory Testing (QST) Procedures. All tests will be conducted in the order described. 
Primary Hyperalgesia. Heat–Pain Threshold(HPTh) will be assessed using an ascending method 
of limits paradigm prior to application of heat capsaicin, after the 35 minute sensitization period and 
after each rekindling. The thermode is secured to the treatment site and the temperature gradually 
increased (rate of rise = .5oC/sec) from a pre-set baseline (31oC), until the subject indicates when 


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the stimulus “first feels painful” (threshold) by pressing a button, which stops and records the 
temperature(98). We will conduct two trials at each QST session, the average of each serves as the 
index of 1oHA(97, 99). 
Secondary Hyperalgesia. The area of 2oHA to mechanical stimulation will be quantified with a 
standard von Frey hair by stimulating along eight linear paths around the treated site in steps of 
5mm at 1 second intervals(96). Stimulation starts well outside the hyperalgesic area and continues 
towards the treated area until the subject reports a change in sensation. The border is marked on the 
skin with a pen and traced to acetate paper. The degree of 2oHA is quantified by calculating the 
surface area using a planimeter (Planix). 
Mechanical Temporal Summation.  Temporal summation of pain will be assessed using 
repetitive mechanical stimuli. The assessment of temporal summation involves rapidly applying a 
series of identical noxious stimuli and determining the increase in pain across trials; animal studies 
have suggested that temporal summation occurs centrally in second-order neurons in the spinal cord 
as a consequence of sustained C-fiber afferent input(100-102).  For temporal summation of 
mechanical pain, pain ratings in response to a single punctate noxious stimulus will be compared to 
pain ratings in response to a sequence of identical punctuate noxious stimuli(103).  A weighted 
pinprick stimulator with a flat contact area will be used to deliver, to the ventral surface of the arm, 
either a single pinprick stimulus or a train of 10 pinprick stimuli repeated at a constant rate. 
Following the single stimulus and the 10-stimulus train, the subject is asked to give a pain rating. 
Single pinprick stimuli are alternated with the trains of 10 stimuli. Similar procedures using this 
type of weighted pinprick stimulator have been used in diabetic neuropathy,(104) healthy 
subjects,(105) and to assess temporal summation(106, 107). 
Pressure Pain Threshold.  An electronic algometer (e.g., Somedic) will be used to assess 
responses to noxious mechanical pressure (108, 109). Pressure is increased steadily at a constant 
rate until the subject indicates that the stimulus “first feels painful”.  
Cold Pressor Pain ToleranceTesting (CPT). Subjects will immerse their capsaicin treated side 
hand in a cold water bath (4oC) for as long as possible, up to an un-informed 5 minute time limit. 
When the sensations become intolerable, subjects remove their hand and the duration of submersion 
is recorded as the index of CPT(110). We decided to include CPT as a primary outcome for Aim 2 
because it is widely used in analgesia studies, and we have found it sensitive to sleep-related effects 
on alfentanil analgesia. It is highly unlikely that CPT will interfere with the heat capsaicin 
procedures as the pain-suppressive effects of such a task return to baseline within approximately 
five minutes(111). 
 
We will perform some of the QST assessments simultaneously. 
 
Optional Sleep, Pain, Emotions, and Reward Study Procedures 
The QST procedures conducted as part of the optional SPEAR study include heat and 
pressure pain threshold, and CPT, as described above.  Additionally, in a separate placebo analgesia 
test, participants will be presented with visual cues (e.g., different colored dots) prior to delivery of 
a thermal stimulus. Subjects will be conditioned to expect more or less pain associated with 
thermode activation based on the presentation of visual cues. The actual temperature of the 
thermode will vary. Subjects will be asked to rate pain intensity and unpleasantness on a 0-100 scale 
in response to the stimuli. Deception is necessary for the effective assessment of placebo analgesia, 
which requires that participants’ expectations be manipulated to compare the difference in pain 
ratings at the same stimulus intensity under different conditions of expectation. Placebo analgesia is 
thought to be regulated by aspects of the reward system, including opioid and dopamine 
neurotransmission, and is therefore an ideal task to include in this substudy, which explicitly seeks 
to understand the range of behavioral reward system responses to sleep deprivation.  


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Participants will consent to “authorized deception” in order to protect their ability to make 
informed consent as much as possible without disclosing the exact moment in which the deception 
will occur, which would invalidate the placebo analgesia task. Participants will be debriefed 
following the completion of the study and informed in explicit terms that the task in which they 
were presented with different colored dots prior to thermal stimulation included thermal stimuli that 
did not, in fact, change in temperature. We will inform them that it was necessary to provide 
misleading information because we are interested in knowing if being sleep deprived alters the 
function of expectation in modulating the experience of pain. We will inform participants that this 
information will further refine our understanding of sleep, pain, and analgesia. Finally, we will give 
participants the option to withdraw their data after learning of the nature of deception. 
 
Drug Protocol. Morphine procedures have been developed based on Dr. Strain’s research and the 
literature(112). No food or non-clear liquids will be permitted for approx. 4 h. (clear liquids 2 h.) 
prior to injection. A CRU pharmacist will prepare the saline or morphine doses onsite. A registered 
nurse (blind to drug condition) will administer the drug via subcutaneous injection. For safety 
reasons, the registered nurse will be aware that the syringe could contain either saline or morphine.. 
Drs. Strain, Tompkins, or Umbricht will be available by pager to provide oversight. 
Physiologic Monitoring and Safety. A nurse will monitor heart rate, blood pressure, SPO2, and 
respiration rate during the procedures. The CRU maintains an anaphylaxis kit, and supplemental 
oxygen. A standing order from Dr. Strain, Tompkins, or Umbricht for Naloxone  will be available 
to fully reverse the effects of morphine in the case of an unlikely untoward event. An Ambu bag is 
also available to mechanically assist ventilation. Due to the effects of sleep deprivation and 
morphine after FA2, subjects will complete a third night of recovery sleep on the unit. To minimize 
burden after the US condition, subjects will be transported home and instructed not to engage in 
certain activities for the next 12 h (e.g., cooking, driving, alcohol). 
 
Inflammatory Mechanisms. Given our preliminary data(113, 114), we will focus on inflammatory 
biology dynamics using a vertically integrated mechanistic approach to examine upstream signaling 
pathways, cellular production of proinflammatory cytokines, and circulating levels. Circulating 
levels and cellular markers of inflammation will be assessed by assaying plasma inflammatory 
biomarkers [(IL-6, TNF-, IL-1ra (surrogate for IL1-β)] in duplicate by ELISA and 
lipopolysaccharide (LPS) stimulated monocyte production of IL-6 and TNF- by flow 
cytometry(113). We will focus on this select set of markers based on our theoretical focus on 
inflammation, our preliminary data, evidence that these markers are associated with pain responses 
in animals(115), impaired morphine efficacy in mice(58), and sleep disturbance(114, 116, 117). 
Because IL1β detection can be unreliable in humans due to low concentrations, we will utlilize IL-
1ra as a surrogate marker for IL-β. For this reason, IL-1ra will be a secondary marker. We will use 
ELISA methods because we have found that multiplex approaches are less sensitive for quantifying 
circulating cytokines(118). Inflammatory signaling will be measured by assay of activation of NF-
B using intranuclear staining and flow cytometric analyses(114). Molecular approaches will 
examine the impact of cytokines on target cell gene expression using highly sensitive RT-PCR 
quantitative analyses of cytokine-regulated gene products in PBMC. We developed and routinely 
perform these approaches at the Cousins Center for Psychoneuroimmunology at UCLA to capture 
the both effects of altered cytokine concentrations as well as changes in cellular sensitivity to 
cytokine activity.  
In vivo cytokine levels. Plasma levels of proinflammatory cytokines from frozen samples will 
be quantified, using Quantikine High Sensitivity human IL-6, TNF-, and IL-1RA immunoassay 
kits (R&D Systems, MN). IL-6 and TNF assays have comparable intra and inter assay coefficients 


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of variation of 4% and 10%, respectively. For IL1ra, the intra and inter assay coefficients are 2% 
and 4%, respectively.  
Intracytoplasmic cytokine expression(113, 119). These assays focus on monocyte cell 
populations as they are the primary source of IL-1, IL-6 and TNF-. For cell preparation and 
intracellular immunostaining of monocytes, we adapted the protocol of Nomura et al.(113). Briefly, 
heparinized blood is aliquoted and co-stimulated with LPS (0.1- 1 (g/ml) to activate monocytes. 
Monocytes are stained with anti-CD14-PerCP and either anti-TNF-PE or anti-IL-6-PE (BDIS). The 
percentage of cytokine-secreting (PE positive) cells among CD14-PerCP positive population is 
determined with counting of 12000 CD14+ events via flow cytometry.  
Molecular Impact of Proinflammatory Cytokines. We have found that mRNA levels for HMG-
1, IRF-4 and IRF-7 are sensitive to IL-1β, IL-6, and TNF- signaling, and are also known to 
increase significantly during in vivo inflammation, thus, serving as sensitive indicators of 
proinflammatory cytokine activity even if cytokine protein levels remain below the limits of 
detection in plasma. To assess changes in indicator gene expression, total RNA samples will be 
extracted from 10 mL of PBMC (Qiagen PAXgene Blood RNA tubes with RNEasy extraction) and 
assayed using the real-time RT-PCR services of the IBC. In this process, total RNA is reverse 
transcribed at 50oC for 30 minutes using target-specific primers (Applied Biosystems Inc. TaqMan 
Gene Expression Assays) in conjunction with the Qiagen 1-step RT-PCR enzyme mix and a BioRad 
iCycler real-time PCR thermal cycler. Following reverse transcription, samples are heated to 95oC 
for 15 minutes to denature the reverse transcriptase and liberate hot-start DNA polymerase, which 
then amplifies reverse transcripts over 40 cycles of universal real-time PCR cycling conditions (15s 
at 95oC, 60s at 60oC). Fluorescent probe signals resulting from DNA amplification are quantified by 
iCycler software and subsequently normalized to GAPDH and β-actin housekeeping genes to 
control for total number of cells assayed. We will use the same approach to assess mRNA levels IL-
1β, TNF-α, IL-6 and their receptors (IL-1R, TNFRII, IL6R, gp160). 
Nuclear Signaling of Proinflammatory Cytokines(114). We have found that sleep restriction 
induces activation of a number of transcription factor families including the pro-inflammatory NF-
B/Rel 235 family. This transcription family has been implicated as critical regulators of gene 
expression in the setting of inflammation. NF-B is clearly one of the most important regulators of 
pro-inflammatory gene expression, and activation of this cellular inflammatory signaling will be 
measured as previously described(120). Briefly, nuclear extracts will prepared from isolated PBMC 
and binding of activated NF-B p65 to its consensus DNA sequence will measured using TransAM 
NF-B p65 ELISA (Active Motif, Carlsbad, CA). In addition, intranuclear signaling will be 
evaluated by flow cytometry for evaluation of resting (i.e., constitutive) and stimulated levels. 
PBMC will be left unstimulated or stimulated for 15 minutes at 37oC with10 ng/ml TNFα for 
induction of NF-B. PBMC will be fixed, permeabilized for nuclear staining, and then incubated 
with phycoerythrin-labeled monoclonal antibodies specific for the active/phosphorylated forms of 
NF-B (p65). Constitutive levels of signaling are determined by mean fluorescence intensity (MFI) 
in unstimulated cells, and the ability of cells to initiate new signaling is determined by comparing 
MFI in stimulated vs. unstimulated PBMC. 
Maintaining Integrity of Assays Between Sites. Using protocols developed by Dr. Irwin, the 
Hopkins RDRC, will receive fresh blood samples from the CRC, prepare them according to 
procedures briefly described above, freeze at -80oC and ship to UCLA for analysis. This includes: 
1) plasma processing; 2) stimulation of whole blood with LPS and fixation for intracytoplasmic 
cytokine expression protocol; 3) preparation of PBMC pellets for isolation of DNA/RNA, and 4) 
PBMC isolation, stimulation with TNF- α, and fixation for intranuclear signaling protocol. To 
ensure fidelity to protocol, Dr. Irwin will travel to Hopkins twice in Year 1 to work with Dr. Soloski 
to train the dedicated RDRC tech. As part of this process, test samples will be performed. All of 
these protocols were developed for the purpose of freezing plasma and cells for future analyses, and 


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include fixation where appropriate to preserve cellular integrity. The protocols are routinely 
performed in Dr. Irwin's laboratory, with frozen samples stored for weeks (cellular assays) to 
months (plasma, DNA/RNA pellets) with excellent results. All samples will be coded and de-
identified to maintain blinding. 
 
Feasibility and Attrition: Since our team has previously executed a similar sleep deprivation 
study, tested opioid protocols, and routinely used quantitative sensory testing, we are confident that 
we can successfully execute the proposed study. In order to maximize retention, our three visit 
screening process and rigorous exclusion criteria minimizes attrition after randomization, ensures 
safety, and minimizes confounds. In our previous sleep deprivation study(34), 20% ruled out at 
screening v1; 10%, at v2, and 5% at v3 (PSG screen). Once randomized, however, only 11% of 
subjects dropped out of the 7 night protocol. The proposed 5 night protocol reduces subject burden 
and may reduce drop-outs. Using these numbers, we plan to conduct: 139 v1s, 111 v2s and 100 v3s. 
We plan to randomize N= 94 and estimate 15% dropout after randomization to achieve 80 full 
completers. To maximize retention, subjects will be remunerated up to $1,050 for their effort ($25 
v1, $25 v2, $150 the PSG screen, $300 for US Condition, $350 for FA condition, and $200 bonus). 
We have included a $200 bonus to incent subjects to return and complete the second admission. In 
our prior study, we recruited 32 female full completers and an additional 12 males in 2 years and 
found no differential drop out between sleep deprivation and control groups. We are, therefore, 
confident that our plans to obtain 80 full completers in 5 years are feasible. 
 
Optional Sleep, Pain, Emotions, and Reward Study Procedures 
Participants who are eligible and elect to participate in the SPEAR study will be compensated an 
additional $300 for daytime testing procedures, including MRI and PET scanning. They may earn 
up to an additional $23.50 based on performance on the motivation and reward tasks (participants 
will not be informed of the total amount they can earn in these tasks in order to minimize 
expectancy effects; however, they will be informed that they can earn some additional money based 
on their performance on the tasks). 
 
b. Study duration and number of study visits required of research participants. 
 
The study will last approximately 5 weeks and involves a total of four visits. 
 
c. Blinding, including justification for blinding or not blinding the trial, if applicable. 
 
This is not a clinical trial. We have adopted a randomized placebo control experimental design, 
however, which requires the subjects and the pain testing technicians to be blind to the subject’s 
group assignment to minimize expectancy effects and technician bias that could comprise the 
causal conclusions from being drawn. A CRU pharmacist will prepare the saline or morphine 
doses onsite. A registered nurse (blind to drug condition) will administer the drug. For safety 
reasons, the registered nurse will be aware that the syringe could contain either saline or 
morphine. 
 
d. Justification of why participants will not receive routine care or will have current therapy 
stopped. 
 
N/A 
 


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e. Justification for inclusion of a placebo or non-treatment group. 
 
Although this is not a clinical trial, the causal nature of the research questions necessitate that 
we control for placebo/expectancy effects.  If subjects were unblinded, it would be difficult to 
draw causal conclusions about whether sleep deprivation alters the effect of opioid efficacy 
because the results would be biased by the subject’s expectancy that pain ratings should 
diminish when given an opioid.  Similarly, if technicians are aware whether a patient is 
receiving morphine, they may inadvertently convey information about how they expect the 
subject to respond.  This would compromise validity. 
 
f. Definition of treatment failure or participant removal criteria. 
 
Participants failing to continue to meet any of the inclusion/exclusion criteria will be removed 
from the study and an appropriate clinical referral provided.  For example, subjects developing a 
psychiatric disorder or symptoms requiring treatment (e.g., mania, suicidality, and psychosis), 
major medical illnesses or serious injuries, which would preclude further participation, will be 
withdrawn.  Subjects who have  severe side effects to morphine will be withdrawn.  Subjects 
who consistently fail to show up for screening sessions will be withdrawn. Subjects who refuse 
the sleep deprivation procedures may be withdrawn. 
5. 
Inclusion/Exclusion Criteria 
 
GENERAL INCLUSION CRITERIA FOR ALL PARTICIPANTS:   
1) Healthy, 18-48 year olds meeting Research Diagnostic Criteria for Normal Sleepers(121); 2) a 
stable sleep phase within 21:00 and 10:00; 3) total sleep time >6.5 and <8.5 hours/night; sleep 
efficiency >85%, Epworth Sleepiness Scale <10; 4) Pittsburgh Sleep Quality Index <5); 5) non-
smokers/nicotine users; 6) low caffeine users (< 2 cups, q.d.). 
 
PET Inclusion Criteria 
1) Have clinical laboratory test results within the reference ranges for the population or results within 
acceptable deviations that are not considered by the investigator to be clinically significant. 
2) All subjects and their partners of childbearing potential must commit to use two methods of 
contraception, one of which must be a barrier method, from the time of screening and throughout 
the study and until follow-up. 
3) Have sufficient venous access. 
 
 
GENERAL EXCLUSION CRITERIA FOR ALL PARTICIPANTS:   
1) BMI >35; 2) lifetime history of chronic pain (>6 
months); 3) acute pain; 4) significant medical or 
psychiatric morbidity within 6 months or lifetime 
history of: bipolar disorder, psychotic disorder, 
serious, recurrent major depression, serious 
posttraumatic stress disorder or seizure disorder; 5) 
respiratory, hepatic, renal or cardiac conditions that 
would contraindicate opioid use; 6) lifetime history 
of alcohol or substance abuse or dependence; 7) lifetime history of opioid use > 36 doses or > 7 
days consecutive use; 8) prior adverse reaction to general anesthetics/opioids or capsaicin; 9) 
clinically significant abnormal complete blood count or comprehensive metabolic profile; 10) 
positive toxicology screen for opioids or recreational drugs; 11) pregnant or lactating women; 12) 
Table 1. Cumulative Enrollment & Scientific Activities 
Year 
Q1  
Q2 
Q 3 
Q4 
1st 
Start up 
Startup 
Active N=5 
N=10 
2nd 
N=15 
N=21 
N=27 
N=33 
3rd 
N=39 
N=45 
N=51 
N=57 
4th 
N=63 
N=69 
N=75 
N=81 
5th 
N=87 
N=94 
Analysis 
Manuscripts 
Analysis 
Manuscripts 


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significant preadmission psychological distress (T-scores >64 on the Brief Symptom Inventory 
global scales); 13) significant lifetime history of serious head injury that is judged to influence pain 
processing or sleep systems. These rigorous criteria are designed to ensure safety and minimize 
major confounds influencing pain, inflammation(122), sleep, or opioid efficacy. We are confident 
of feasibility based on our prior work with similar criteria(34, 123). See enrollment plan, Table 1. 
 
PET Exclusion Criteria 
1) Have participated in other research protocols in the last year such that radiation exposure would 
exceed the annual limits. 
2) Pregnant or nursing women. 
3) History of head trauma with prolonged loss of consciousness (>10 minutes) or any neurological 
condition including stroke or seizure (excluding childhood febrile seizure) or history of migraine 
headache. 
4) History of any clinically relevant hematological, hepatic, respiratory, cardiovascular, renal or CNS 
disease or other medical condition that is capable of constituting a risk factor when taking the study 
drug. 
5) Abnormal vital signs, ECG or clinical laboratory evaluations which are considered clinically 
significant by the clinical investigator. 
6) Suffer from claustrophobia and would be unable to undergo MRI or PET scanning. 
7) Clinically significant abnormal MRI.  
8) Subject has implanted or embedded metal objects, prostheses, or fragments in the head or body that 
would present a risk during the MRI scanning procedure, or have worked with ferrous metals either 
as a vocation or hobby (for example, as a sheet metal worker, welder, or machinist) in such a way 
that might have led to unknown, indwelling metal fragments that could cause injury if they moved 
in response to placement in the magnetic field. 
 
 
Rationale for Eligibility Criteria. The normal sleeper profile is typically used in insomnia 
research (e.g.,(124) and must be evident both at intake (retrospective) and as an average profile (2-
weeks of baseline diaries and actigraphy)).  The RDC criteria for normal sleep rules out individuals 
meeting criteria for sleep disorders, including sleep apnea and periodic limb movement disorder, 
etc. The Visit 3 PSG screening will be used in the diagnostic process to rule individuals with occult 
sleep disorders based on standard ICSD criteria using objective PSG derived indices of sleep 
disordered breathing (respiratory disturbance index <15 and apnea/hypopnea index <10), periodic 
limb movements, etc.  Because we are seeking to experimentally determine the effects of sleep 
deprivation on hyperalgesia, individuals with sleep disorders are precluded from participation as 
this would confound the findings. 
Adults over 48 are excluded because of age-associated reductions in slow wave sleep(125), 
increased risk of sleep disorders, and comorbid medical factors might confound results. Reduced 
slow wave sleep has been shown to influence pain sensitivity(126).   
Severely obese individuals with BMIs above 35 are excluded because they are at high risk for 
exclusion, due to sleep apnea and other co-morbidities (127).  
Seizure disorder, bipolar disorder, and psychotic disorders are ruled out because sleep 
deprivation may exacerbate these conditions. Similarly, we require subjects to report psychological 
distress within normal limits because sleep deprivation is a stressor, which could unduly impact an 
individual with very high levels of distress. Individuals with a significant psychiatric disorder 
within the past 6 months are excluded because these conditions can be associated with prolonged 
sleep architecture changes even with symptom remission. 


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Respiratory, hepatic, renal, or cardiac problems, which would either significantly alter morphine 
metabolism or which could be detrimentally impacted by morphine effects are exclusions. 
Morphine lowers respiratory drive and therefore can pose significant hazard to individuals with 
conditions such as asthma.  
Individuals with alcohol and substance abuse disorders are excluded because opioids have 
significant abuse potential. We are studying relatively opioid naïve subjects because opioid 
exposure and long-term use can alter opioid receptor sensitivity and regular use can lead to 
tolerance. 
Restrictions on nicotine and heavy caffeine usage are required due to known effects on pain 
testing and/or inflammation.  Heavier caffeine usage can cause withdrawal symptoms when 
discontinued, which could confound hyperalgesia testing. These criteria are commonly used in the 
pain testing literature and we have not found them to pose a recruitment problem(93). 
A normal CBC is required because infections, etc can significantly alter sleep and sleep need, 
which might skew results. 
PET-related eligibility criteria is designed to minimize risks associated with MRI and PET 
imaging, as well as to ensure that the brain-based comparisons across subjects are not influenced by 
abnormal neurological profiles. 
 
6. 
Drugs/ Substances/ Devices 
 
a. The rationale for choosing the drug and dose or for choosing the device to be used. 
Selection of Capsaicin, Dose, and Administration.  Capsaicin was chosen because its application in 
the context of a continuous thermal probe provides a unique opportunity to measure both primary 
and secondary hyperalgesia in a manner that cannot be as effectively accomplished without its 
application. The coupling of heat with capsaicin permits the study of primary and secondary 
hyperalgesia without causing tissue damage.  Further, it uniquely affords the opportunity for 
multiple rekindlings of thermal pain over time, which is a crucial element of the experimental 
design.  The dose chosen (0.1% Capsaicin) has been previously validated (85) and safely used in 
other studies from our laboratory. 
 
Selection of Morphine, Dose (.08 mg/kg), Blinding, and Administration. We considered a variety of 
opioid agonists, doses, and routes of administration. We selected morphine for several reasons: 1) it 
is the prototypical mu opioid receptor agonist without clear sex differences in efficacy(81, 128); 2) 
prior animal studies of cytokines blocking opioid analgesia used morphine(57, 58, 129, 130) and 3) 
morphine is efficacious in reducing 1o and 2o HA in the heat capsaicin model(131). We selected the 
.08 mg/kg dose, which is lower than some controlled studies(132), to minimize possible side-
effects, which might increase drop out and because several studies demonstrate efficacy of a single 
.08 mg/kg dose on a wide range of laboratory pain tests, including CPT(92, 133). In consultation 
with the CRC, we decided to administer the drug via subcutaneous injection. 
 
Selection of radioligand for PET imaging. The current protocol employs an IND for [11C]raclopride 
(IND # 32,914).  The sponsor- investigator, Dr. Dean Wong, has had the raclopride IND since 1989, 
and the IND was more recently amended to include AMP.  [11C]raclopride is now widely employed 
throughout the world as a radiotracer for measuring dopamine D2 receptors with high specific 
activity (radiotracer alone) and low specific activity (added non-radioactive mass saturates some of 
the receptors).  More recently, however, [11C]raclopride has been used to measure intrasynaptic 
dopamine release following various perturbations; specifically, high specific activity raclopride 
radiolabels D2 receptors, but can be competitively displaced by endogenous intrasynaptic dopamine.  
Dopamine release into the synapse, in turn, can be modulated by psychological challenges such as 


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cocaine craving cues, video games, etc (Drobes and Tiffany, 1997; Wong et al, 2006).  Thus, 
[11C]raclopride provides a unique tool for measuring not only D2 receptors (high and low specific 
activity), but also for measuring the intrasynaptic dopamine tone (high specific activity alone). 
 
b. Justification and safety information if FDA approved drugs will be administered for non-FDA 
approved indications or if doses or routes of administration or participant populations are changed. 
N/A 
 
c. Justification and safety information if non-FDA approved drugs without an IND will be 
administered.  
N/A 
 
 
 7. 
 Study Statistics  
 
a. Primary outcome variables. 
 
Selection of Independent Variables and Primary Outcomes. The primary independent variable in 
all analyses is the within-subject comparison of SD versus US.  We will also evaluate whether 
sleepiness (SSS), fatigue (diary), positive and negative affect (PANAS-X) and spontaneous pain 
(diary) are associated with outcomes and include these variables as covariates in secondary analyses 
when indicated. Primary outcomes were chosen based on evidence from our prior work and the 
literature, described above.  A summary of primary outcomes by aim is presented in Table 2.  
  
 
 
 
 
 
 
 
 
b. Statistical plan including sample size justification and interim data analysis. 
Analytic Plan  Hypothesis 1.a.(Sleep disruption enhances HA): We will use multilevel longitudinal 
mixed effects models to test the null hypothesis that baseline and sleep-deprived indices of 1o & 2o 
hyperalgesia are equal. We will also test for differences in linear time trends by condition.  
Hypothesis 2.a. (Sleep disruption diminishes morphine analgesia): Similar models will test the 
null hypothesis of no effect of sleep deprivation on the analgesic effects of morphine on 
hyperalgesia and cold pain tolerance. We will also test for differences in linear time trends. 
 
Hypothesis 3.a. (Sleep disruption increases activation of markers of inflammation): We will first 
compute area-under-the-curve-ground (AUC-G) and -increase (AUC-I) values following standard 
formulas(134) for the inflammatory markers. AUC allows us to simplify the analysis of the repeated 
measures data to increase power(134). AUC-G represents the total amount of inflammation during 
the pre-drug administration segment (blood draws1-4), whereas AUC-I represents the level of 
increase in inflammation in response to heat capsaicin (blood draws 3-4), relative to the within-
condition baseline levels (blood draws 1 & 2). Mixed effect models will test the difference in 
marker AUCs between sleep conditions. 
Table 2: Summary of Outcome Measures By Aim 
AIM 
CONSTRUCT 
MEASURE 
Aim 1-3 
1oHA♦ peripheral sensitization 
2oHA♦ spinal sensitization 
1oHA-Heat pain threshold (oC), treated skin 
2oHA-Surface Area (cm2), mech. HA untreated skin 
Aim 2 & 3 
Pain Tolerance 
Duration (sec) of hand submersion in 4oC Water 
Aim 3  
Inflammation  
- Systemic 
- Cellular 
- Genomic 
- Genomic Signaling 
 
Circulating (plasma) IL-6, TNF-α , IL-1RA** 
Stimulated & Unstim. PBMC levels of IL-6 & TNF 
PBMC mRNA conc. of IL-6**, TNF-α**, IL-1β** 
Stimulated & Unstim. PBMC levels of NF-B 
♦ Heat Capsaicin Testing; ** secondary outcomes 


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Hypothesis 3.b. (Markers of inflammation mediate the effects of sleep disruption on: 1) HA and 
2) diminished opioid analgesia):  Following the MacArthur approach(135) we will first establish 
relationships between the markers and both hyperalgesia (HA) and sleep disruption (SD). Thus, 
these analyses will only be conducted if tests of Hypothesis 3.a. indicate a relationship between SD 
and activity of a specific marker. For these markers, we will then test the relationship between 
marker activity and HA using mixed effects models. When significant, interactions between SD and 
marker activity will be taken as evidence of mediation rather than moderation, as should a main 
effect of marker activity after controlling for this interaction and the main effects of SD(135).These 
analyses use only data from the pre-drug administration segment ;therefore, the placebo and opioid 
groups are combined. Mixed effects models will test the relationship between marker activity and 
diminished opioid analgesia, allowing for correlation within individuals across sleep conditions and 
pre- and post-drug segments. Statistical significance of the main effect of marker activity, the 
interaction between drug condition and marker activity, or the interaction between drug, marker, 
and SD will be taken as evidence of a mediating effect of marker activity.  
 
Power Analysis & Sample Size.  We calculated sample sizes via Monte Carlo simulation(136, 
137). Continuous variables were standardized to express regression coefficients as effect sizes. 
Power for a given sample and effect size was calculated as the percentage of datasets (1000 
simulations) for which either: 1) the p-value for a wald test or 2), the p-value for a likelihood ratio 
test for nested models was lower than the alpha specified for the model. Based on the simulations 
described below, we determined that a sample size of N=80 will be adequate to accomplish all 
of our Aims. 
Aim 1: (Sleep disruption enhances HA). To account for correlation between measurements 
within individuals across sleep conditions, a parallel process model was parameterized for 
MPLUS(136, 137) with the baseline mean outcome set to 0 and the SD mean outcome set to a range 
of values representing various effect sizes. Linear trends within sleep condition are not 
anticipated(96, 97), so slopes (and variance of slopes) were simulated as 0. With an N=80, we 
estimate 80% or greater power for effect sizes of 0.40 or greater. In our preliminary heat-capsaicin 
pilot (see section 4.A.1.), poor sleep was associated with an effect size of 0.57 on 2oHA. This 
correlational data, in a naturalistic sample of healthy subjects, is likely to underestimate the effect of 
our sleep disruption condition on HA. 
Aim 2: (Sleep disruption diminishes morphine analgesia). Similar to Aim 1, a parallel process 
model (this time with 4 processes, one per segment and sleep condition) was parameterized for 
MPLUS. As above, all slopes and random slope variances were simulated as 0. The difference in 
drug effect as a function of sleep condition was based on Steinmiller’s study of the effect of codeine 
on daytime sleepiness (effect = 0.9)(138) and our pilot data showing the effects of poor sleep on 
alfentanil vs. placebo (see 4.A.2; effect size = 1.9). Since the hypothesis test involves 4 separate 
parameters, power was calculated via a likelihood ratio test with 1 degree of freedom comparing an 
unconstrained model to one in which the parameter function was constrained to be 0. Simulations 
showed that with an N=80, we have 80% power for effects of 0.7 or greater. Therefore, we are 
sufficiently powered to detect similar effects reported in the literature and in our pilot data.   
Aim 3: (Sleep disruption increases activation of markers of inflammation). Because several 
inflammatory markers are being investigated, we will employ a more conservative alpha value of 
0.01. This simulation was parameterized similar to Aim 1, but with main effects of both marker 
activity and sleep deprivation and their interaction and with a single mean outcome for each sleep 
condition. With N=80, we have 80% power to detect main effect sizes for marker activity of >0.56. 
Based on our prior work (see section 4.A.4), with the exception of the mRNA markers (effects =.2 
to .36), the effects for 1 night of sleep restriction on all other markers range from .56 (cellular 


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production of IL-6/TNF) to .76 (NF-B). Because our sleep manipulation is much more robust (two 
nights of FA), we expect to be well powered to detect effects on all inflammatory markers. To be 
conservative however, and to further reduce multiple comparison error, we will consider the mRNA 
markers as secondary outcomes. 
 
c. Early stopping rules. 
We will report all adverse events and evaluate on a case-by-case basis whether the occurrence of a 
particular adverse event has implications for discontinuing the study to ensure participant safety and 
well-being. 
 
8. 
Risks 
 
a. Medical risks listing all procedures, their major and minor risks, and expected frequency. 
 
Listed below are all major procedures and associated risks. All risks are determined to be minimal.  
Please refer to the Method section for detail.  All of these standardized procedures have been 
conducted with participants both in earlier studies from our laboratory, and in other laboratories 
around the world. 
  
Administration of Morphine (.08 mg/kg).  Morphine is an opioid receptor agonist, which has high 
affinity for mu receptors, and is widely used in the treatment of moderate to severe pain. Previous 
literature has demonstrated the effects and side effect profile of the current route and dose of 
morphine(133). We are using a routine dose based on body weight that is lower than is commonly 
use in emergency departments to treat acute musculoskeletal pain(139). This substantially 
minimizes possibility of toxicity and reduces potential side effects. 
Major risk factors of morphine toxicity are respiratory depression (diminished respiratory 
drive), and possible death. Additional serious side effects are anaphylaxis, seizures, falling or 
unsteadiness, confusion, unusual changes in mood or behavior, slow heart rate (bradycardia), 
difficulty passing urine, hypotension, severe constipation, slowed cognition, and psychomotor 
impairment. 
Minor risks and the most common side effects include: nausea, vomiting, lightheadedness, 
dizziness, euphoria, sweating, constipation, sleepiness, and pruritis, and mild allergic reaction(s) 
(i.e., rash, hives, itching, wheezing).  
 
Sleep Disruption/Deprivation: The most common risks associated with sleep deprivation include:  
sleepiness, poor concentration, slowed reaction times, irritability, negative mood, and a diminished 
capacity to operate a motor vehicle or perform work. Sleep deprivation lowers seizure threshold, 
and rarely, may trigger manic episodes in individuals with bipolar disorder.  
 
Topical Capsaicin: Capsaicin is the pungent ingredient of hot chili peppers and has been contacted 
and ingested by the vast majority of individuals. In some cultures it is consumed in large quantities 
without serious risk. Our group has tested well over 100 subjects using topical capsaicin at this 
dose, combined with heating the skin at the temperatures proposed and with some subjects 
undergoing administration of capsaicin 4 times over the course of a protocol. During these hundreds 
of administrations, there have been no serious adverse events. The most common adverse reactions 
are application site erythema and pain, which is usually mild to moderate. Pruritus is also common. 
Less frequently, temporary papules may develop. Intense pain can be experienced if capsaicin 


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comes into contact with mucous membranes or compromised skin (open sores/wounds).  Mild 
sensitivity to warm/hot temperate at the site of application may persist for a couple days. 
 
Heat-Pain Testing: There is an extremely small chance that the heating device might produce a 
burn, but the risks associated with the relatively small amount of heat applied to the skin (maximum 
of 122 degrees Fahrenheit or 50 degrees Celsius) are very low. The Medoc device has safety 
features built into both the hardware and software. The risks associated with use of this equipment 
are less than those associated with common household appliances. We and others across the world 
have previously used the proposed laboratory pain procedures in a number of previous studies with 
healthy adults and chronic pain patients. No serious adverse effects were reported. 
 
Mechanical Temporal Summation: There is a very slight chance that the probe might superficially 
puncture the skin for individuals with “thin skin.” 
 
Pressure Pain Threshold: There is a very slight chance of mild transient bruising associated with the 
use of an algometer (< 5% of cases). 
 
Cold Pain Tolerance Task: There are no significant risks from immersion of a hand in cold water by 
a healthy individual for the 5 minute maximum time permitted.  The task could exacerbate 
Reynaud’s syndrome, therefore these patients are excluded. 
 
Intravenous Catheterization/Blood Draws: Some discomfort may be associated with the drawing of 
blood samples, and there is a very small chance that phlebotomy may cause hematoma, infection, 
anemia, and/or minor pain at the puncture site. However, these procedures are routinely performed 
without incident in medical settings, and catheters will be installed by qualified nursing staff. 
 
Subcutaneous Injection: Some pain or discomfort around the injection site may be associated with 
the injection of drug or placebo.  
 
Saline Placebo: There are no risks associated with saline itself. Minimal risks associated with 
subcutaneous injection are described above.  Injections will be administered by qualified nursing 
staff. 
 
Polysomnography (Sleep Monitoring): There are minimal risks associated with routine 
polysomnography testing. Very rarely, individual may experience a mild allergic reaction to 
electrode gels/paste. 
 
Physical Exam and Structure Diagnostic Interviewing: There are no known or reported medical 
risks associated with undergoing a physical exam or the proposed diagnostic interviews performed 
by a qualified professional. 
 
Urine Sample: There are no risks to collecting a sample of urine.  Strict confidentiality of the 
sample & results will be maintained. 
 
Actigraphy: There are no risks associated with wearing a wrist actigraph. 
 
Questionnaires: Minimal risks associated with completing the questionnaires are subject fatigue and 
the possibility of minor psychological distress associated with answering sensitive questions 
regarding psychological functioning. 


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Optional Sleep, Pain, Emotions, and Reward Study Procedures 
Emotion-Related Tasks:  Some of the images presented during the emotion-related tasks are 
graphic, including images of violence, mutilation, death, and erotica. The graphic nature of these 
images is similar to what is permitted in R-Rated movies. It is possible that some participants may 
be distressed after being exposed to these images. However, the risks associated with these images 
are expected to be minimal based on the fact that they have been standardized on thousands of 
individuals and included in hundreds of research studies conducted with similar populations. 
 
Electromyography and Galvanic Skin Response Monitoring: There are minimal risks associated 
with routine electromyography and galvanic skin response testing. Very rarely, the individual may 
experience a mild allergic reaction to electrode gels/paste. 
 
Motivation, Reward-Related, and Psychomotor Vigilance Tasks: There is a minimal risk that 
participants may experience fatigue and/or boredom while completing some of these tasks. 
 
Additional Pain Testing: See risks described above under Heat Pain Testing, Pressure Pain 
Threshold, and Cold Pain Tolerance Task. 
 
Placebo Analgesia:  The primary risk associated with this task pertains to deception. 
Research deception may cause: 
1. Infringement of the autonomy of research subjects  
2. Distress and lack of trust in research  
3. Negative emotional reactions 
 
MRI: MRI does not involve exposure to ionizing radiation and has no known serious risks. 
Individuals with pacemakers, aneurysm clips, shrapnel or other unallowed implanted metallic 
devices will be excluded from study.  All subjects complete the standard MRI screening 
questionnaire prior to MRI study. Participants may experience claustrophobia before entering the 
MRI machine and during the performance of the MRI while lying in the MRI machine.  If 
participants are markedly bothered by claustrophobia, they will be eliminated from the study.  
Participants may experience fear and discomfort during the MRI due to the noise of the machine.  
To decrease the noise of the MRI, participants will be asked to insert ear plugs before entering the 
MRI. 
 
Skull x-ray. As indicated in the MRI procedures, participants may be asked to undergo two skull x-
rays to determine the participant has not metal in their skull.  This is a standard safety procedure 
employed by the Johns Hopkins MRI staff to verify it is safe for the participant to enter the MRI 
area.  If participants undergo the skull x-ray there will be a radiation exposure of 0.01 rem to the 
participant. 
 
Radiation: As [11C]raclopride is a radiotracer, participants will be exposed to radiation.  The amount 
of radiation exposure to a participant in this protocol will be approximately 0.94 rem. This total 
radiation exposure is within allowable radiation exposure limits (not to exceed 5 rem per year). 
 
Radiotracer: [11C]Raclopride will be administered during the two scans with high specific activity 
[11C]Raclopride. The risks of adverse pharmacological events are negligible.  Less than 15µg will 
be administered for each scan. The risks associated with much higher levels (therapeutic) may 
include transient sleepiness, restlessness, mild muscular spasms or dystonias (more severe but still 


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transient muscular spasms). The administered dose is lower than the usual therapeutic dose which 
should make the risk of getting any of the above mentioned side effects less likely. Should any of 
these side effects occur they are easily treatable and reversible. Tardive dyskinesia, a sometimes 
irreversible syndrome involving involuntary movements of the face, mouth, or other parts of the 
body, is an uncommon side effect typically seen in patients taking Raclopride or similar drugs for a 
prolonged period of time. At all times, participants will be monitored by a physician who will take 
appropriate measures to remedy these rare effects. 
 
PET scanner. Participants may experience discomfort while lying on the PET scanner table for the 
scans.   Participants will be repositioned and cushioned with padding if they feel discomfort. 
 
Thermoplastic facemask. The thermoplastic facemask may cause the participant some discomfort. If 
the mask is too tight, we will remold it or reposition the participant. They may experience some skin 
irritation when the facemask is created. 
 
b. Steps taken to minimize the risks. 
 
All personnel involved in study procedures will be fully trained in the protection of human subject 
as required by our IRBs. If information gathered during either study indicates that an individual is in 
need of emergent psychiatric care (e.g., expressed suicidality). Drs. Smith, Buenaver, and Campbell 
are licensed psychologists, and one of the three will immediately assess the needs of the participant 
who will be referred for psychiatric care at Johns Hopkins or an appropriate community referral. 
During any phase of the study, subjects who report psychological distress related to their 
participation will be provided a referral to a mental health professional who will provide a one-
session consultation. Described below are specific protections against risks by procedure. 
 
Administration of Morphine (.08 mg/kg).  As described, individuals with respiratory (e.g., asthma), 
hepatic, renal, cardiac, or other medical conditions that would contraindicate morphine 
administration will be excluded. Blood tests verifying liver and kidney function will be conducted 
and reviewed by one of the study physicians (Dr. Strain,Tompkins or Umbricht) prior to 
implementing the procedure. Subjects with a history of allergic reaction to opioids or anesthesia are 
excluded. We are also excluding individuals with alcohol and substance abuse disorders because 
opioids have significant abuse potential. An appropriately licensed CRU pharmacist will prepare the 
morphine doses onsite. A registered nurse will administer the drug as prescribed by Dr. Strain, 
Tompkins, or Umbricht. For safety reasons, the registered nurse will be aware that the syringe could 
contain either saline or morphine. The subject will remain in a recumbent position during the 
procedure to prevent falls.  Drs. Strain, Tompkins, or Umbricht will be available by pager to 
provide any medical oversight as needed. A nurse will monitor heart rate, blood pressure, SPO2, 
and respiration rate during the procedures. The CRU maintains an anaplylaxis treatment kit and a 
supplemental oxygen tank. Sufficient naloxone will be available onhand. An Ambu, CPR bag is 
also available to facilitate mechanical ventilation if needed. The CRC also has an AED. The 
protocol will include a standing naloxone order signed by Dr. Strain, Tompkins, or Umbricht 
sufficient to fully reverse the adverse effects of morphine should a subject experience respiratory 
distress or other morphine-related complications. Nurses will be trained under the direction of Drs. 
Strain and Tompkins. The CRC has established a standard emergency protcol to ensure the safety of 
subjects undergoing opioid administration.We are using a routine dose of morphine based on body 
weight that is typically lower than is commonly use in emergency departments to treat acute 
musculoskeletal pain(139). This substantially minimizes possibility of toxicity and reduces potential 
side effects. Drs. Strain and Tompkins are both very experienced physicians who specialize in and 


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routinely care for patients with opioid dependence. They have considerable expertise with clinical 
and research protocols involving opioid administration and naloxone/naltrexone blocade. Dr. 
Umbricht is also very experienced in these domains and will be available as study physician in the 
event that both Drs. Strain and Tompkins are unavailable. Subjects receiving morphine under sleep 
deprivation conditions will be required to remain on the Unit for a recovery period (overnight) prior 
to disharge to prevent accidents due to impairment caused by morphine plus sleep deprivation. After 
the undisturbed sleep condition, subjects will return home the same day, but will not be permitted to 
drive and instructed not to engage in potentially hazardous activities for the next 12 hours, e.g., 
cooking, driving, operating heavy machinery, or drinking alcohol or other depressants. 
The nursing staff is well trained to assist subjects experiencing side effects such as nausea and 
emesis. As described, food and drink is limited and restricted prior to injection to minimize these 
side effects. Subjects will complete the 6-item drug effect VAS and an adjective checklist (36 
items), after injection to gather data on frequency and severity of side effects. Dr. Strain has used 
these measures in his opioid pharmacology work to study side-effects of opioid medications. This 
will be reported regularly to the IRB and our data safety and monitoring committee for review. 
These measures were specifically designed to capture common and uncommon side effects of 
morphine and opioid medications. 
 
Sleep Disruption/Deprivation: Because sleep deprivation lowers seizure threshold and may trigger 
manic episodes in individuals with bipolar disorder, individuals with these conditions are excluded 
as described. All subjects undergoing sleep deprivation will remain under surveillance on the 
medical research unit for a recovery night sleep to prevent accidents that might be caused by 
drowsiness. Because sleep deprivation is a stressor that can impact mood, we have excluded 
subjects with major psychopathology or acute psychological distress. 
 
Topical Capsaicin: Prior to capsaicin application, we will ensure that the skin is not compromised at 
the site of administration. Participants are instructed that they may discontinue any of the pain 
procedures at any time should they become too intense or unpleasant. Ice will be available for 
application, which dramatically reverses the heat-capsaicin pain if subjects wish to discontinue. 
Further, when the protocol is finished, we will cover the application area with a large band-aid and 
give participants several additional band aids so they can keep the area covered in order to avoid 
accidentally rubbing it on any other bodily areas that could be potentially painful (e.g., rubbing their 
eye(s), mucous membranes).   
 
Heat-Pain Testing: All pain testing equipment will be tested prior to use to ensure it is functioning 
properly. Johns Hopkins IRB requires that all devices are approved by bioengineers for safety and 
proper functioning before approval of any research protocol. Thermodes and devices touching 
participant’s skin are sterilized before and after use with an appropriate agent. The thermal sensory 
analyzer has built-in safety features to prevent burning. The temperature to be used will not cause 
burn. For all of the standardized laboratory pain procedures, participants are repeatedly informed 
that they may terminate any of the procedures at any time, and participants are monitored 
continuously by the technician. In the event that a subject is injured in any way by any of the study 
procedures, Dr. Tompkins, Strain, or Umbricht will evaluate the condition and, if appropriate, refer 
the participant for medical attention and notify the IRB, as appropriate. Across hundreds of research 
participants with various conditions, we have had no adverse events deemed greater than mild (even 
this level AE occurs in < 1/100 cases) related to the use of these devices.  
 


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Cold Pain Pain Tolerance Task: There are no significant risks from immersion of a hand in cold 
water by a healthy individual for the 5 minute maximum time permitted.  The task could exacerbate 
Reynaud’s syndrome, therefore these patients are excluded. 
 
Blood Samples. There is a very small chance that phlebotomy may cause hematoma, infection, 
anemia, and minor pain at the puncture site. However, these procedures are routinely performed 
without incident in medical settings, and catheters will be installed by qualified nursing staff 
according proper sterile technique.  
 
Subcutaneous Injection: Subcutaneous injection is routinely performed without incident in medical 
settings. This route of administration was selected over intramuscular injection because it is better 
tolerated by patients (140). A qualified nursing professional will administer the subcutaneous 
injection according to proper techniques. 
 
Saline Placebo: There are no risks associated with saline itself. Minimal risks associated with the 
routine IV catheterization procedure are described below.  Catheters will be installed by qualified 
nursing staff. 
 
Polysomnography testing. Very rarely, individuals may experience a mild allergic reaction to 
electrode gels/paste. The CRU has appropriate medicine (e.g., cortisone cream) to ameliorate 
discomfort as needed. 
 
Physical Exam and Structure Diagnositic Interviewing. There are no known or reported medical 
risks associated with undergoing a physical exam or the proposed diagnostic interviews performed 
by a qualified professional. 
 
Urine Sample. There are no risks to collecting a sample of urine.  Strict confidentiality of the 
sample & results will be maintained. 
 
Actigraphy. There are no risks associated with wearing a wrist actigraph. 
 
Questionnaires. Minimal risks associated with completing the questionnaires and cognitive testing 
are subject fatigue and the possibility of minor psychological distress associated with answering 
sensitive questions regarding psychological functioning. Subjects are instructed that they do not 
need to disclose any information that may be too uncomfortable.  A Certificate of Confidentiality 
has been obtained to further protect participant privacy and ameliorate subjects’ concerns regarding 
sensitive information. 
 
Emotion-Related Tasks: Participants will be made fully aware of the types of graphic content 
included in the International Affective Picture System images at the point of consent, and then again 
prior to engaging in the task.  They will have opportunities to decline participation or discontinue 
participation at any time. Potential participants with severe or recurrent psychopathology will not be 
eligible to participate, so it is unlikely that participants will become severely distressed as a result of 
viewing these images. 
 
Electromyography and Galvanic Skin Response Monitoring: Very rarely, individuals may 
experience a mild allergic reaction to electrode gels/paste. The CRU has appropriate medicine (e.g., 
cortisone cream) to ameliorate discomfort as needed. 
 


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Motivation, Reward-Related and Psychomotor Vigilance Tasks: To mitigate the unlikely potential 
for excessive fatigue and/or boredom, subjects will be reminded that they may discontinue any task 
at any time. 
 
Additional Pain Testing: See risk minimization steps described above under Heat Pain Testing, 
Pressure Pain Threshold, and Cold Pain Tolerance Task.  
 
Placebo Analgesia:  
Risk 1: Infringement of participant autonomy. This risk is sensitively minimized when prospective 
subjects are given an “authorized deception” disclosure that alerts them to the use of deception in 
the research during the informed consent process. We inform research participants about the use of 
deception, the fact they have the opportunity to decline to participate, and that they will learn the 
specific nature of deception at the end of their study participation.  
 
Risk 2: Distress and lack of trust in research. Revealing the truth about the study during the 
debriefing process may potentially result in participant’s distress who can be upset at learning how 
they were deceived. However, based on literature on deception in placebo research and our 
experience, no lack of trust in research have been observed. 
 
Risk 3: Negative emotional reactions. No negative reactions or lasting negative consequences from 
learning the details regarding the use of deception have been observed. We believe this has been 
mitigated by the authorized deception part of informed consent. 
 
From the perspective of researchers, the authorized deception may influence the placebo-induced 
analgesia by reducing beliefs and certainty about the treatment. This point deserves future research. 
 
 
PET and MRI procedures: Before and during PET scans, the physiological and psychological status 
of each participant will be closely followed. Before and during each PET scan, blood pressure, heart 
rate, and rhythm strip will be carefully monitored.  
 
A physician will monitor the physiological and psychological status of the participants before, 
during, and after inventions until the participant returns to the baseline level. The physician will 
institute the appropriate medical and psychological interventions when they are needed.  
 
If an acute clinical problem is encountered in the course of the study, the participant will be referred 
for treatment as indicated. 
 
c. Plan for reporting unanticipated problems or study deviations. 
 
Any protocol deviations and/or adverse events will be systematically documented by Dr. 
Michael T. Smith, the PI, and reported immediately to the Johns Hopkins IRB. Case Report 
Forms (CRFs) will identify study participants by their initials and a unique study participant 
identifier. The participant’s name will not be attached to any data. CRFs will be stored in a 
locked office, which is only accessible to the study coordinator and authorized personnel. The 
principal investigator will provide an interim report of all adverse events to the IRB at the time 
of continuing review. 
 


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The FDA and IRB will be notified of serious adverse events associated with [11C] raclopride, in 
accordance with the FDA and IRB reporting requirements. 
 
All adverse events, unanticipated problems, or study deviations will be reported per the current 
JHM IRB guidelines pertaining to problem/event reporting posted on the JHM IRB website. 
 
d. Legal risks such as the risks that would be associated with breach of confidentiality. 
 
Some of the personal health information gathered during the screening process or during treatment 
is sensitive.  The laboratory blood testing will identify the presence of illegal drugs (if detectable), 
which might have consequences if they were released to others. Every effort will be made to 
maintain privacy and confidentiality.  A number of procedures will be in place to prevent a breach 
of confidentiality. All potential subjects will be fully informed of their rights pertaining to 
disclosure of PHI in accordance with HIPAA regulations. The proposed research will be subject to 
the strict rules of confidentiality maintained at Johns Hopkins Medical Institutions. Confidentiality 
will be maintained by assigning participants a study number and numerically coding all data. One 
hard copy file linking the code number with identifying information will be kept in separate locked 
file with direct access available to the PI only. All records and research data will be kept in locked 
filing cabinets or computers.  Information about urine toxicology screening results will be handled 
by Dr. Smith and the project coordinator and will be kept separately from the study clinical record 
in a locked file maintained by Dr. Smith.  Only summaries of group data will be reported in any 
publications or presentations, with no identification of individuals. These precautions should serve 
to minimize legal risks to participants. The PI is a psychologist, licensed in the state of Maryland, 
and is bound by state laws on confidentiality.  Further, Johns Hopkins is committed to protecting 
the rights of potential research participants.  In light of this, the Hopkins IRB has developed a 
training program on protection of human subjects that is required for all Hopkins personnel engaged 
in research involving human subjects.    
e. Financial risks to the participants. 
 
There are no financial risks or costs to study subjects for any of the procedures. 
 
9. 
Benefits 
a. Description of the probable benefits for the participant and for society. 
 
All participants will receive, at no cost, a comprehensive evaluation of their sleep and mental health 
status, including a screening polysomnographic sleep study, which might identify an occult sleep 
disorder such as sleep apnea.  These individuals will be informed of any disorders identified and 
referred for appropriate clinical care.  Other than mentioned above, there is no other benefit to 
participation for subjects in the study, but the risks to participation are minimal and justifiable.   
   
10. 
Payment and Remuneration 
a. Detail compensation for participants including possible total compensation, proposed bonus, and 
any proposed reductions or penalties for not completing the protocol. 
 
Subject Compensation 
Visit 1  
 
 
 
 
=      $25  
Visit 2   
 
 
 
 
=      $25 
PSG  Screen  
 
 
 
=      $75 
US Condition  
 
 
 
=    $300 


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Page 27 of 42 
 
FA Condition  
 
 
 
=    $350 
Completion Bonus       
 
 
=    $275  
Total                       
 
 
 
= $1,050 
 
Subjects will be remunerated up to $1,050 for their effort ($25 v1, $25 v2, $75 the PSG screen, 
$300 for US Condition, $350 for FA condition, and $275 bonus). We have included a $275 bonus to 
incent subjects to return and complete the second admission. For participants who leave the study 
early for any reason, they will receive partial compensation pro-rated as a function of their 
participation. 
 
Optional Sleep, Pain, Emotions, and Reward Study Procedures 
Participants who are eligible and elect to participate in the SPEAR study will be compensated an 
additional $300 for daytime testing procedures: $100 for each PET session, $25 for each QST and 
reward testing session, and a $50 completion bonus. 
 
Thus, the total amount a participant could earn for participation if they are eligible and participate in 
all procedures associated with the Mechanisms of Sleep Disruption Hyperalgesia study and the 
SPEAR study is $1,350.  In addition to the participation payments, participants may earn a small 
amount of additional money (up to $23.50) as part of the motivation tasks. This amount will not be 
advertised or included in screening or consenting because it could create expectancy effects that 
could bias results.  However, participants will be informed that they can earn some additional 
money based on their performance on the tasks. 
 
11. 
 Costs 
a. Detail costs of study procedure(s) or drug (s) or substance(s) to participants and identify who will 
pay for them. 
 
 
There are no costs to study participants. 
 
 
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