Population and subject recruitment

This single site, ongoing, IRB-approved pilot study (Clinicaltrials.gov registration NCT0323089), is being carried out in the Department of Neurology at the Wake Forest School of Medicine, Winston-Salem, North Carolina, USA. Initial eligibility screening is conducted through an online questionnaire followed by a phone conversation. To be considered for inclusion, individuals must be active duty military service members or recent veterans with service since 2001 with symptoms of military-related traumatic stress, including insomnia, poor concentration, sadness, irritability, or hyper-alertness, with or without a history of traumatic brain injury (TBI). Participants are required to have either a formal diagnosis of PTSD, a referral from a military medical provider confirming active PTS symptoms, or prior or current treatment for the same. For those participants who are special operations service members, the study deliberately does not use a symptom inventory threshold score as an eligibility criterion because of the under-reporting of symptoms among these individuals (personal communication, Naval Special Warfare medical officer). If contact is established through self-referral with the absence of a formal PTSD diagnosis, a score of 50 points on a screening PCL-M is required. Potential participants have been identified by referrals from military medical providers, as well as the Care Coalition and Preservation of the Force and Family, which both support the special operations community of the United States Armed Forces. Several participants have joined through self-referral after word of mouth from other participants or review of open studies on the research program webpage on the Wake Forest Baptist Health website. Based on advice from military personnel and recognition that the potential stigma associated with a diagnosis of PTSD might limit recruitment, study flyers and related materials focused on symptoms and did not include use of the term “PTSD.”

Exclusion criteria are the inability to provide informed consent, inability to attend all study visits or sit comfortably in a chair, bilateral total hearing loss, known seizure disorder, or an ongoing need for the use of benzodiazepines, opiates, anti-psychotic medications, selective serotonin reuptake inhibitor (SSRIs) or selective norepinephrine reuptake inhibitor (SNRIs), prescribed sleep medications including zolpidem or eszopiclone, stimulant medication, or thyroid hormones. Those with ongoing or anticipated regular use of recreational drugs, alcohol, or energy drinks during the intervention and in the 4 weeks following intervention completion or a lack of internet or smart phone access were also excluded. With the knowledge of and under the direct management of their medical provider, participants could titrate off what would otherwise have been considered exclusionary medications or recreational substances prior to enrollment.

Intervention schedule

Beginning on a Monday morning, and following informed consent, baseline (Visit 1, V1) outcome measures are collected (details below), including self-reported symptom inventories, physiological and functional measures, an assessment of brain electrical activity, blood and saliva samples for biomarkers or epigenetic testing, and a whole brain, resting-state MRI scan. Participants then receive a series of closed-loop acoustic stimulation sessions (HIRREM) over a period of 12 days. The initial two sessions are given on the afternoon of the first day following the completion of all baseline data collections. Thereafter, participants receive two sessions daily, with a break between sessions. Typically, no sessions are given on Saturday (day 6), many receive a single afternoon session on Sunday (day 7), with a final, single morning session on the second Friday (day 12), prior to the repeated outcome measures.

All outcome measures are repeated prior to departure on day 12 (Visit 2, V2), except that for scheduling purposes and to ensure similar time of day of sampling, blood and saliva collection followed the morning session on day 11. Symptom inventories are collected remotely via online surveys at 1, 3, and 6 months following intervention completion (V3, V4, V5, respectively). Brief informal interviews are conducted with participants in person during V2 data collection, and narrative comments are sought at subsequent data collections by either phone or email.

Outcome measures

Symptom inventories

A panel of outcome measures evaluate clinical symptoms related to PTSD, insomnia, depressive mood, and anxiety. The Posttraumatic Stress Disorder Checklist, military version (PCL-M) measures the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) Criteria B, C, and D for PTSD symptoms based on traumatic life experiences related to military service [31]. Seventeen items are rated on a Likert scale with a composite score range of 17 to 85. A score of 50 or higher is correlated with a probability of military-related PTSD [32], although cutoff scores as low as 30 to 34 have been suggested for active-duty soldiers seen in primary care populations [33]. A reduction of ≥ 10 points in the PCL-M has been suggested to be a clinically significant change [34]. The Insomnia Severity Index [35] is a 7-question measure, with responses from 0 to 4 for each question, that yields scores ranging from 0 to 28. A score of 15 or greater is considered to indicate moderate or greater insomnia severity, and 8 to 14 indicates subthreshold insomnia. A reduction of at least 6 to 7 points has been suggested as the minimally important clinical difference for insomnia symptom reduction [36, 37]. The Center for Epidemiologic Studies Depression Scale (CES-D) [38] is a 20-item survey assessing affective depressive symptomatology to screen for the risk of depression. Scores range from 0 to 60, and a score of 16 or greater is commonly used as a clinically relevant cut-off [39]. The Generalized Anxiety Disorder 7-item scale (GAD-7) [40] is a seven-item screening tool for anxiety that is widely used in primary care. The clinical threshold to consider treatment is 8, and a statistically reliable change is 4 or greater. Subjects with a history of mild traumatic brain injury or concussion also complete the Rivermead Post-Concussion Questionnaire [41], a 16-item survey that assesses the severity of common post-concussion symptoms on a scale of 0 to 4, with a total score range from 0 to 64 (least to highest symptom severity).

Autonomic cardiovascular regulation

Continuous recordings of blood pressure (BP) and heart rate (HR) are obtained from noninvasive finger arterial pressure measurements and electrocardiogram for 10 min with subjects resting supine and breathing freely. These recordings follow the completion of the symptom inventories and functional testing. Systolic, diastolic, and mean arterial BP, as well as beat-to-beat RR intervals (RRI) files generated via the data acquisition system (BIOPAC acquisition system and Acknowledge 4.2 software, Santa Barbara, CA) at 1000 Hz are analyzed using Nevrokard SA-BRS software (by Nevrokard Kiauta, d.o.o., Izola, Slovenia). All recordings are visually inspected, and the first 5 min of usable tracings are analyzed. Recordings with dropped beats or gross motion artifacts are excluded from analysis. Assessments included multiple measures of heartrate variability (HRV) in both time and frequency domains, baroreflex sensitivity (BRS), and blood pressure [42].

Functional testing

Reaction testing uses a drop-stick, clinical reaction time apparatus. It is constructed from a meter stick covered in friction tape with gradations. The modified meter stick is fixed to a weighted rubber cylinder. The apparatus is placed between the thumb and index finger of the subject and released at a random time during a countdown. The subject catches the apparatus and the distance it has fallen is measured. Following two practice trials, subjects perform 8 trials, and the mean distance value is used for analysis [43]. Grip strength evaluation is done using a hydraulic hand dynamometer (Baseline Hydraulic Hand Dynamometer). The greatest force generated during three trials is used for analysis [44].

Biomarkers of stress and inflammation and epigenetic measures

During the study, funding became available to permit limited exploratory analysis of post-interventional changes in markers of stress and inflammation in 15 subjects and for epigenetic measures in 8 subjects. Blood-based measures included Angiotensin II (Ang II), Angiotensin 1 to 7 (Ang 1–7), epinephrine, norepinephrine, C-reactive protein (CRP), vasopressin, Interleukin 1 (IL-1), Interleukin 6 (IL-6), and Interleukin 10 (IL-10), and saliva measures included cortisol and alpha-amylase. For epigenetic testing, DNA was isolated from whole blood samples to quantify DNA methylation at each site. Microarray assays were used to determine the methylation proportion for each site (beta value) based on the ratio of the fluorescence intensity of the methylated versus the combined methylated and unmethylated probes.

Closed-loop allostatic neurotechnology intervention

The process and procedures for the provision of closed-loop allostatic neurotechnology by a technologist in an office setting have been discussed in detail previously [16]. An initial assessment of brain electrical activity entails two-channel recordings from at least 6 paired locations on the scalp (F3/F4, C3/C4, T3/T4, P3/P4, FZ/OZ, O1/O2; also, typically FP1/FP2 and CB1/CB2) with the participant at rest and while carrying out a task, using sensors and amplifiers that sample at 256 Hz. At each scalp location, data are recorded for 1 minute each with eyes closed, eyes partially open as a transitional state of arousal, and eyes open while carrying out a specific mental task (e.g., reading numbers or performing mental calculations). Trained technologists evaluate assessment data to choose protocols for the initial intervention session.

Protocols for each session include recording brain electrical activity through, generally, two channels, with scalp sensors placed at homologous regions of the hemispheres according to the 10–20 International EEG system. Software algorithms analyze specific ranges of the brain electrical frequency spectrum in real time, identify dominant frequencies based on proprietary mathematical formulae, and translate those frequencies into acoustic stimuli (audible tones of variable pitch and timing) which are presented to participants through standard earphones (Creative EP-630 or Sony Stereo Headphones MDR-EX58V) with as little as an eight-millisecond delay. Volume (decibels) of acoustic stimulation is adjusted for each participant in accordance with their preference.

Each session (typically 90–180 min each) consists of 4 to 10 protocols, ranging from 5 to 40 min per protocol, and each is intended to address a specific anatomical location and frequency range. Some protocols are completed with eyes closed and some with eyes open, with the participant being asked to relax while sitting or reclining comfortably in a zero-gravity chair. After the initial session, specific protocols and protocol durations for successive sessions are chosen based on brain electrical data from the participant’s preceding session, which, for purposes of technologist review, are aggregated in broad-band frequency ranges (< 1.0 Hz; 1.0–3.0 Hz; 3.0–5.5 Hz; 5.5–7.5 Hz; 7.5–10.0 Hz; 10.0–12.0 Hz; 12.0–15.0 Hz; 15.0–23.0 Hz; 23.0–36.0 Hz; 36.0–48.0 Hz). Special attention is given to activity set points suggestive of dominant hemispheric asymmetries and/or suboptimal ratios of energy across the frequency spectrum. Algorithms are designed to support the de-establishment of relatively invariant and potentially maladaptive activity patterns. All participants continued with their current medical or behavioral care being used at the time of enrollment.

Although exact mechanisms await confirmation, it appears that with rapid updates regarding its own electrical activity, intended to support frequency-matching or resonance between the acoustic stimulation and oscillating brain networks, the brain is supported towards auto-calibration and self-optimization. As a closed-loop process, no conscious or cognitive activity is required, yet the brain pattern is observed to shift on its own terms towards improved balance and, often, reduced hyperarousal.

Statistical analysis

A repeated-measures ANOVA was performed to evaluate changes in symptom inventory scores between baseline and each follow-up visit. For other comparisons, two-tailed paired t-tests were performed to evaluate pre- to post-HIRREM changes. In consideration of the sample size, the non-parametric Wilcoxon signed-rank test was used to corroborate the t-test findings. Analyses were performed using SAS (Cary, NC).