Study Hypotheses and Aims:
The primary hypothesis of the INVICTA study is that exposure to repeated subconcussive blast events during training with shoulder-fired heavy weapons (e.g., recoilless rifles, shoulder-mounted assault weapons, and anti-tank rockets), as quantified through the use of personally worn blast sensors, will be associated with acute and longer-term changes in the serum levels of UCH-L1, a marker of neuronal injury. The secondary hypothesis is that exposure to RSCBE during training with shoulder-fired heavy weapons, as quantified through the use of personally worn blast sensors, will be associated with changes in recall memory function. Exploratory aims are to, 1) explore the relationship between extent of blast exposure and clinical [i.e., neurocognitive, neurobehavioral, neuromotor (including oculomotor assessments), neurosensory, autonomic, vision, and hearing function] and pathophysiological (i.e., markers of neuronal, glial, vascular and blood-brain barrier damage, inflammatory and immune system reaction) outcome domains, 2) evaluate changes in acute responses to blast exposure of range safety officers (RSOs)/instructors at the start vs. end of their 2-year tour of duty, 3) explore potential subject-specific effect modifiers (factors that may alter subject response to blast exposure), 4) explore the efficacy of alternative measures of blast exposure (e.g., maximum overpressure, positive phase duration, and frequency of exposure) and related factors (e.g. type of shoulder-mounted weapon and ammunition) to predict and/or modify outcome, and 5) assess the impact of repetitive blast exposure on neuroimaging parameters (e.g., number of white matter hyper intensities, resting state functional connectivity, fractional anisotropy), by imaging RSOs both at baseline and 18–21 months later.
INVICTA is an observational, prospective, longitudinal study featuring both within- and between-subject comparisons to better define the magnitude of RSCBE and to document the consequent cellular, neurocognitive, physiological and functional changes. Eligible participants included Special Operators and range safety officers (RSOs, or instructors) participating in HWT [i.e., firing the Carl Gustav recoilless rifle, the Light Anti-Armor Weapon (LAAW), and the AT-4 anti-tank rocket], or who are going through the same training but fire training rounds or other weapons that are found not to result in significant pressure recordings on blast gauges (training controls), and non-Operator, Active Duty SMs who are not going through training and do not have blast exposure (naïve controls). The independent variable is RSCBE, measured by blast gauges mounted on helmets, shoulders and chests of participants. Outcome assessments were taken at baseline (prior to firing for exposed participants) and at 30 minutes, 6, 24 and 72 hours, and 2 weeks (acute response), 3 months (subacute) post-firing ̶ or associated time points for controls ̶ for all participants, and 9 and 18 months (chronic, accompanied by a repeat of the acute and subacute response assessments (RAR) over the ensuing 3 months after the 18-month time point), for RSOs. This study design allows for outcome assessments to be compared with baseline for within-subject comparisons, identifying changes in assessments between the pre-exposure baseline and the post-exposure time points, and for between-subject measures between the exposed and the two control groups.
INVICTA has a target population of 300 participants: 100 Special Operators who fire weapons of interest during HWT; 50 Special Operators who take part in HWT but do not fire weapons of interest and do not have peak blast overpressures of > 2.5 psi recorded on their blast gauge sensors; 100 Range Safety Officers (RSOs), and 50 active duty military service members who are not Special Operators. Prior to the conduct of any assessments, informed consent is obtained from each participant by either the principal investigator or other study staff who have proven their ability to administer consent to the satisfaction of the principal investigator. The consent process includes providing the participant with a consent form that conveys detailed information about any benefits and risks, the voluntary nature of participation, and the ability to withdraw from participation at any time. This information is also conveyed verbally, time for participants to ask questions is provided, and participants are queried to confirm their understanding of key points. Both the participant and the administrator of the consent sign the form, and a copy of the signed consent is provided to each participant.
Self-Reported Military and Health Status
Participants complete each of the following:
Demographic questionnaire (e.g., marital status, age, race, ethnicity, education level, military history);
Basic medical history ̶ including caffeine, alcohol, tobacco, medications, and supplement use. Administered at baseline, updated as needed at subsequent assessments.
The Ohio State University TBI Identification Method (OSU TBI-ID), a reliable and valid structured interview that documents lifetime TBI history, administered at baseline by a trained research team member, and updated as needed at subsequent assessments.
The following questionnaires are administered at baseline and 3 months for all participants, and at 9, 18 and 21 months for RSOs.
Patient Health Questionnaire-9 (PHQ-9) measure of depression symptom severity.
PTSD Checklist for DSM-5 (PCL-5) measure of PTSD symptom severity.
General Anxiety Disorder (GAD-7) measure of anxiety symptom severity.
Neurobehavioral Symptoms Inventory (NSI) measure of post concussive syndrome symptom severity.
Veterans RAND 12-Item Health Survey (VR-12) measure of overall functional status and quality of life.
Headache Impact Text (HIT-6) assessment of the impact of headaches on the ability to function at school, work, home, and in social situations.
Pittsburgh Sleep Quality Index (PSQI) measure of sleep quality, sleep disturbances, and sleep-related functioning.
Pain is assessed using the Defense Veterans Pain Rating Scale (DVPRS) at every time point, including the use of Supplemental Questions at all time points except 30 min and 6 hours after blast exposure.
The Black Box Biometrics (B3) Generation 7 (Gen 7) Blast Gauge System (BGS) is utilized to measure blast overpressure. The BGS is a collection of three watch face-size devices (mounted on the back of the helmet, on the non-dominant shoulder and chest of the uniform or body armor); each device contains a pressure measurement sensor, an accelerometer, and a microprocessor. The microprocessor is programmed to identify potential blast-related events based upon changes in pressure measurements and, when such an event is detected, to record the pressure and acceleration until blast-related energy dissipates (approximately 20 milliseconds). Applied Research Associates (ARA) cleans the data, removes artifacts and adjusts for the angle of the blast, and then generates 3D blast biophysical reconstructions from the BGS overpressure waveforms. Study participants are issued gauges at the time of baseline assessment. Participants are shown how to place the gauges on their helmet, chest, and non-firing shoulder; how to “awaken” the gauges by moving them; and how to check for cumulative overpressure exposure by pressing the recessed activation button. They are instructed to wear the gauges when participating in training events in which there is a potential for exposure to blast overpressure. Overpressure and acceleration data are downloaded from the gauges by a USB cable connected to a laptop computer at all data collection time points after the index training exposure. The data are then transferred to the Center for Neuroscience and Regenerative Medicine (CNRM) CASA data platform, where the data can be accessed by ARA for post-processing and analyses. We also have study staff observe and take notes on the type of weapon fired, and the position and distance that the participant is from the weapon, and this is recorded on a Manual Firing Line log, which also incorporates input from the study participant.
Blood is collected at every time point by study personnel who are trained phlebotomists following Standard Operating Procedures (SOPs). The venipuncture procedure is standard, consisting of the participant’s blood collection into two 10.0 mL Serum Separating tubes (SST) and a single 8.5 mL PAXgene DNA tube. The whole blood in PAXgene DNA tube requires no further processing beyond a room temperature incubation period for a minimum of two to maximum of 48 hours. Post incubation, the PAXgene tube is then inventoried and flash-frozen as appropriate. The SSTs are processed by activating the clotting agent and allowing 30–45 minutes for incubation at room temperature. Post incubation, SSTs are centrifuged for 10 minutes at 1100–1300 rcf. The participant’s serum is aliquoted in 400µl quantities into identification specific cryogenic vials. These vials are then inventoried and stored in 81 grid cryoboxes and flash-frozen as appropriate. PAXgene tubes and serum samples are processed, flash-frozen as appropriate, and shipped to the CNRM Biorepository in Bethesda, MD. Individual identified samples will be logged in, catalogued using the specimen management system of the CNRM, and maintained in -80 degrees Celsius freezers. INVICTA serum samples are analyzed in duplicate using the ultra-sensitive Single Molecule Array (SIMOA) Assay (Quanterix, Lexington, MA) for measurement of tau, neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and Ubiquitin C-Terminal Hydrolase L1 (UCHL-1) (Human Neurology 4-Plex A assay (N4PA), Cat# 102153) concentration on an HD-X Simoa instrument according to instructions from the manufacturer (Quanterix, Billerica, MA). Serum samples are measured in SIMOA HD-X within machine 4-fold dilution. Briefly, four distinct, dye-encoded bead populations are presented with analyte-specific capture antibodies that are first incubated with samples and biotinylated detector antibodies. The target molecule present within each sample are captured by capture beads and labeled with the corresponding detector antibodies. The bead-conjugated immunocomplex is thoroughly washed and labeled with streptavidin-conjugate b-galactosidase. Following a final wash, resorufin b-D-galactopyranoside is added. The bead-conjugated immunocomplexes are loaded on the SIMOA array disc, which is designed to enable imaging of each bead via their encoded dyes and fluorescent substrate generated signals. The number of bead-containing wells producing positive signals is proportional to the number of target molecules within the sample for each plex. The average number of enzymes per bead (AEB) of each sample fit into a four-parameter logistic curve plotted using the known concentration of the calibrators. The correlation is confirmed for the accuracy of fit and for the conversion of AEB values to concentrations. The average coefficient of variation of biomarkers are highlighted if above 30%. The primary biomarkers of interest are ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), neurofilament-light (NF-L), tau and glial fibrillary acidic protein (GFAP).
The primary method for the assessment of neurocognitive performance is with the Hopkins Verbal Learning Test – Revised (HVLT-R). This assessment is a list learning test that consists of 12 nouns within three semantic groups using six alternate forms that evaluates verbal learning and memory. The administrator reads 12 words aloud, with an interstimulus interval of two seconds, and the participants are asked to immediately recall them. The list is read a second time followed by a second immediate recall. The list is then read a third time followed by a third immediate recall. The words recorded for each recall trial are then tallied to calculate the total recall score of all three trials. The participants are then informed that the examiner will ask them to remember the trial words at a later time during the visit. A delayed recall trial begins 20–25 minutes after the completion of the third immediate recall trial, during which time participants are engaged in other assessments; the participant is asked to recite from memory as many of the words on the list as possible. A percent retention is then calculated by dividing the total number of target words from the delayed recall trial by the highest score of the second or third immediate recall trial, multiplied by 100. Following the delayed recall trial is a yes/no delayed recognition trial which consists of 24 words (12 true-positive words from the original list, interspersed with 6 semantically related false-positive words and 6 semantically unrelated false-positive words). The examiner reads each word and asks the participant to respond with ‘yes’ if they believe the word was on the original list or ‘no’ if they believe it was not. The Recognition Discrimination Index is scored by calculating the total number of true-positive words minus the total number of semantically related and semantically unrelated false-positive words. The HVLT-R is administered at baseline, 30 minutes, 24 hours, 72 hours, 2 weeks, and 3 months for all participants, as well as 9 and18 months, plus a full RAR assessment series, for RSOs.
In addition to the HVLT-R, The Automated Neuropsychological Assessment Metrics Version 4 (ANAM-4), a validated, widely used, computer-based tool designed to detect speed and accuracy of attention, memory, and thinking ability, is also utilized. The ANAM can assess processing speed, attention span, memory, and other cognitive components. The full ANAM Concussion Protocol battery is administered at Baseline, 72 hours, 18 months, and the 18 month RAR 72 hour time point. At every other time point except for the 6 hour and 18 RAR 6 hour, at which the ANAM will not be administered, only the ANAM Reaction Time and Matching to Sample tests will be administered.
Auditory function, which is frequently a concern related to blast exposure, is assessed with a tablet-based hearing assessment known as TabSINT, an open source platform for administering tablet-based hearing related tests as well as questionnaires. Each tablet is paired with the Wireless Automated Hearing Test System (WAHTS) from Creare LLC. The easily portable WAHTS provides the capabilities of a fully functional audiometer and connects to the Android tablet running TabSINT via a Bluetooth connection. This system enables calibrated threshold-level audiometric measurements via the TABSINT platform. The highly attenuating earcups (or headphones) of the system provide noise isolation comparable to a single-wall booth . The INVICTA audiologic assessment includes detection and discrimination tasks. For example, the participant is asked to use a button to detect the presence of a tone or speech signal presented over headphones (the Bekesy Threshold Measurements and Audiometry Fixed-Level Frequency Thresholds), or the participant hears an audio signal presented in conjunction with white noise and is asked to use response buttons to identify which sound is heard (Triple Digit Test and Binaural Masking Level Difference Test). Self-report hearing surveys are also administered to assess hearing complaints reported before and after blast, including The Hearing Screening tool (THS) and the Acute Auditory Change Questionnaire (AACQ). The THS is a validated assessment that measures the impact of tinnitus, hearing loss, and sound tolerance on a 0 (No, not a problem) to 4 (Yes, a very big problem) point scale . The AACQ is used to assess hearing ability before and after blast or noise exposure .
A more detailed battery is delivered at baseline and 18 months for Range Safety Officers (RSOs), which includes the Full AACQ, Bekesy Threshold Measurements for frequencies from 0.5-8k Hz, Audiometry Fixed-Level Frequency Thresholds, Triple Digit Test, and Binaural Masking Level Difference Test. A shortened battery, consisting of 2 AACQ questions per ear, Bekesy Threshold Measurements for 4 and 6k Hz only, the Triple Digit Test, and the Binaural Masking Level Difference Test is administered 30 minutes, 6 hours, 2 weeks and 3 months after blast exposure, as well as at 9, 18 and 21 months for RSOs.
INVICTA conducts standard structural MRI, resting state functional MRI and DTI, in RSOs at baseline when they begin their 2-year tour as a Land Warfare training RSO, and again 18–21 months later, near the end of their tour. RSOs typically are exposed to blast over the course of 10–12 Heavy Weapons Training exercises during the intervening months. Participants are screened by study staff prior to the scan to ensure that they do not have shrapnel or other ferro-magnetic metal in their bodies that could pose a risk in the scanner. The MRI protocol employs commercially available and widely published pulse sequences, including DTI, DWI, gradient recalled echo (GRE), T1 MPRAGE, T2-weighted fluid attenuated inversion recovery (T2-FLAIR) and resting state fMRI.
Neuromotor performance is assessed with the AccWalker, a valid and reliable  smartphone-based dynamic balance test that utilizes the phone’s to objectively measure neuromotor performance. This neuromotor assessment uses a stepping-in-place task as a dynamic challenge to the balance control system . A smartphone is placed on the participant’s thigh and the AccWalker measures the thigh’s motion throughout the task. Participants are asked to step in place with eyes closed for one 30-second practice trial and two 70-second trials. Several variables are derived from the thigh motion time series that characterize temporal and spatial aspects of the movement (e.g., time between strides, range of motion of the thigh, and variability in motion between strides). The AccWalker is administered at baseline and 6 and 72 hours, 2 weeks and 3 months for all participants, as well as at 9, 18 and 21 months for RSOs.
Oculomotor, vestibular, reaction time, and cognitive (OVRT-C) performance is measured with the Dx-100, a portable, 3D head-mounted display system with integrated eye tracking technology. This FDA-cleared device provides visual and auditory stimuli and records the horizontal, vertical, and torsional movement of the eye, along with pupillary changes, and also records the participant’s visual and auditory reaction time. Participant are asked to wear the 3D head-mounted display system and complete various oculomotor, vestibular, and reaction tests following stated instructions. The Dx-100 is administered at baseline, 24 hours, 2 weeks, and 3 months after blast exposure, as well as at 9, 18 and 21 months for RSOs.
The Lucid™ M1 System Transcranial Doppler (TCD) measures cerebral vascular reserve in the Middle Cerebral Arteries (MCA) to calculate a vaso-motor reactivity (VMR) index. With participants in the supine position, a bilateral TCD monitoring exam is conducted where two transducers are simultaneously applied to the temporal region targeting the right and left MCA to record the mean velocity, pulsatility index (PI) (PI = peak systolic velocity-diastolic velocity/mean velocity), and vaso-motor reactivity index (VMR Index = 100% x (Velocity_EndC02 - Velocity EndHyper/Velocity Baseline) with breath holding and hyperventilation. This VMR monitoring exam includes a 30 second baseline recording, 30 second breath holding, 30 second recovery period, and then 30 seconds of hyperventilation. The TCD assessment is conducted at baseline, 6 hours, 72 hours, and 3 months for all participants, as well as at 9, 18 and 21 months for RSOs.
Possible blast exposure-related changes in tactile neurosensory functioning are examined using the custom-built two-point vertical displacement stimulator Brain Gauge™ (Cortical Metrics, LLC) device. The somatosensory system has been suggested to be highly sensitive to brain injury-related changes in function. The organization of the somatosensory system enables the study of cortical–cortical interactions in adjacent or near-adjacent cortical regions using a tactile interface device that measures the variability in human reaction to tactile stimuli. Alterations in sensory perception occur in parallel with alterations in systemic cortical alterations. The Brain Gauge device utilizes gentle mechanical stimulation (vibration) via computer-generated sinusoidal waveforms that are relayed to mechanical transducers that stimulate the fingertips. The vibrations) are delivered at varying amplitudes (0-300 microns) at constant frequency (25Hz) for short durations (less than or equal to 750 msec), via a flat Delrin probe (5–10 mm in diameter) positioned to make contact with the index and middle finger of one hand (D2 and D3). The device is low-powered and requires only a single connection via USB with the computer or laptop that is used to administer the test(s), which makes it particularly useful in the remote site trailers in which it is administered for this study. The stimulator can apply single site or two-site stimulation to digits two or three at specified amplitudes or frequencies. Tests can be conducted to assess reaction time, amplitude discriminative capacity, frequency discriminative capacity, temporal order judgment, timing perception, and effects of conditioning stimuli on discriminative capacity. Data are collected as quantitative values of neurosensory perceptual metrics and are automatically stored. A full Brain Gauge Assessment is administered at Baseline and 2 weeks post shoot; partial assessment is completed at all other time points.
Elements of the Brain Gauge test battery include the following:
Reaction Time (RT) [1, 19, 56–62] Participant receives a single pulse to the middle finger; participant clicks the mouse button as soon as they feel the pulse. There is no on-screen button to click, clicking anywhere on the screen will record their reaction time and progress the trial.
Amplitude Discrimination (AD) [19, 56–66] Participant compares two vibrations. Vibrations will be delivered sequentially or simultaneously to the left (index) and right (middle) fingers. Participant is directed to pick the larger / stronger / more intense vibration by clicking on the left or right button on the screen. Response time is not important for this task, allowing the participant to make the best choice possible.
Temporal Order Judgement (TOJ) [19, 57, 60, 66–69]. Participant receives two pulses – one to each finger - with one preceding the other. The participant is asked to indicate “Which one came first?” The participant responds using the USB mouse to indicate LEFT or RIGHT on the screen of the computer. The participant should pick the “first stimuli” by selecting the right or left button on the screen of the computer.
Duration Discrimination (DD) [19, 57, 60, 62, 63, 66]. Participant receives two vibrations of different durations. One vibration is delivered to one digit, and after a brief pause the other is delivered to the adjacent digit. Participant indicates which stimuli was longer.
Electroencephalography (EEG) is obtained through the use of the BrainScope One™, a handheld brain injury assessment device that uses digital EEG technology. Raw patterns of EEG signal are recorded by the device and the manufacturer’s proprietary brain function index values provide an EEG-based Brain Function Index that compares the participant’s brain function to an age-regressed normed population, expressed as a percentile (0-100). Quantitative EEG testing is performed at Baseline, 6 hours, 24 hours, 2 weeks and 3 months to all participants. In addition, it will be administered to RSOs at 9 months and at the 18 RAR at 6 hours, 24 hours, 2 weeks and 3 months.