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Research Report

Havana Syndrome: Neuroanatomical and Neurofunctional Assessment in


Acquired Brain Injury Due to Unknown Etiology
Submitted by:
Alon Friedman, MD, PhD1
Cindy Calkin, MD, CCFP, FRCPC1, 2
and
Chris Bowen, PhD3
Brain Repair Center, Dalhousie University and Nova Scotia Health Authority
Halifax, Nova Scotia.
May 24th, 2019

Co-Investigators: Ori Brenner, BvSc4; Laine Green, MD5; Rishi Gupta, MD6; Erez Hanael , DVM7;
Javeria Hashmi, PhD8; Jong Sung Kim, MS, PhD9; Robert Laroche, MD6; Diane MacKenzie10, PhD;
Darren Oystreck, PhD11; Greg Noel, AuD12; Matthias Schmidt, MD13; Maher Quraan, PhD3; Derek
Rutherford, PhD14; Merav Shamir, DVM7; Janine Verge, AuD12

Research Assistants and Students: Amanda Adams, BSc1, 2; Guillermo Artisti Suarez, PhD8; Kathleen
Cairns, BSc1,2; Lyna Kamintsky, MSc1; Jillian Newton, BSc1; Crystal Sweeney, MSc8, Margaux Ross,
MA1,2; Claire Reardon, BSc1,2.

1
Department of Medical Neuroscience, Faculty of Medicine, Dalhousie University, Halifax, NS
2
Department of Psychiatry, Faculty of Medicine, Dalhousie University, Halifax, NS.
3
Biomedical Translational Imaging Centre (BIOTIC), Halifax, NS.
4
Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel
5
Department of Neurology, Faculty of Medicine, Dalhousie University, Halifax, NS.
6
Department of Ophthalmology and Visual Sciences, Faculty of Medicine, Dalhousie University,
Halifax, NS.
7
Veterinary Neurobiology, Koret School of Veterinary Medicine , The Hebrew University of
Jerusalem, Israel.
8
Department of Department of Anesthesia, Pain Management and Perioperative Medicine,
Dalhousie University, Halifax, NS.
9
Health and Environments Research Centre (HERC) Laboratory, Department of Community Health
& Epidemiology, Faculty of Medicine, Dalhousie University, Halifax, NS.
10
School of Occupational Therapy, Faculty of Health, Dalhousie University, Halifax, NS.
11
Clinical Vision Science, Faculty of Health, Dalhousie University, Halifax, NS
12
Nova Scotia Hearing and Speech Centres, Halifax, NS.
13
Department of Diagnostic Radiology, Faculty of Medicine, Dalhousie University, Halifax, NS.
14
School of Physiotherapy, Faculty of Health, Dalhousie University, Halifax, NS.
1
Brain Repair Centre
Dalhousie University | Life Sciences Research Institute | 1348 Summer Street, North Tower | Halifax NS B3H 4R2
www.brainrepair.ca


I Executive Summary:
Background: Since Autumn 2016, US and Canadian government personnel and their families in
Havana began presenting with a variety of neurological manifestations suggested to result from a
brain injury of unknown etiology.
Approach: We report on a study of 28 participants, including 12 “non-exposed” individuals who
never lived in Havana, 11 “recently exposed” - who were tested within one month of returning from
Havana, and 14 “remotely exposed” –tested 1-19 months after returning from Havana (median 14
months). Seven individuals were tested twice, before and after 5-7 months of being posted in
Havana.

Participants were interviewed to obtain medical history, completed self-reported symptom


questionnaires and computerized cognitive assessment (CANTAB). Blood samples were collected
for complete blood count, routine biochemistry, kidney, liver, and metabolic functions. Serum was
obtained for biomarker analysis. Brain imaging included magnetic resonance imaging (MRI)
sequences for fiber tractography and for the detection of blood-brain barrier (BBB) dysfunction;
magnetoencephalography (MEG) was used to assess changes in brain activity. Individuals with
suspected evidence of brain injury underwent further neurological, visual, movement, auditory and
vestibular assessments.

Results: In contrast to previous reports on American diplomats 1,2, most Canadians did not describe
an acute, directional, unusual sensory and/or auditory stimulus, but rather a gradual development of
debilitating symptoms. Similar to previous descriptions by American diplomats, most common
symptoms included a general feeling of impaired wellbeing (sleep disturbance, fatigue, headache and
irritability), poor cognition (concentration and memory), visual disturbance (blurred vision and
sensitivity to light) and audio-vestibular symptoms (tinnitus, sensitivity to sound and dizziness). No
significant differences were found between recently and remotely-exposed individuals.
Anthropmetric measures and routine blood test results were typical of that for the Canadian
population, with no differences between the groups. Self-rated questionnaires were consistent with
“post-concussion syndrome” and disabling headaches. Anxiety, depression and post-traumatic stress
disorder were uncommon. Cognitive tests showed a significant reduction in spatial memory and a
milder decrease in decision making quality in both exposed groups. Visual assessment was
unremarkable. Auditory assessments revealed no hearing loss or major disability. Brainstem evoked
potentials, acoustic reflex and myogenic response to auditory stimuli were positive in 60-80% of
exposed individuals, suggesting brain-stem dysfunction. Routine clinical MRI was unremarkable.
Diffusion Tensor Imaging (DTI) and fiber tractography revealed a significant reduction in fiber
density in the fornix and posterior part of the corpus callosum in exposed groups. Dynamic contrast
enhanced (DCE)- MRI showed a significant increase in BBB permeability in six non-overlapping
brain regions in individuals scanned after exposure compared to pre-exposure scans. A significant
increase in regional BBB dysfunction was found in the right basal forebrain and anterior insula in
exposed compared with non-exposed controls. MEG analysis confirmed a significant increase in the
occurrence of ‘slow wave episodes’ in exposed individuals, particularly in the recently-exposed
group, compared with non-exposed controls. Histopathological post-mortem brain examination of a
neurologically-injured exposed dog affected while with his owners in Havana confirmed a brain stem
lesion with white- and gray-matter BBB leakage and reactive gliosis.

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Dalhousie University | Life Sciences Research Institute | 1348 Summer Street, North Tower | Halifax NS B3H 4R2
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The clinical presentation, cognitive impairment in the spatial memory domain, positive
auditory-vestibular results, degradation of fiber tracts in the fornix on DTI, leaky brain vessels
on DCE-MRI and abnormal brain slowing on MEG all support the diagnosis of acquired brain
injury in the Canadian diplomats and their families posted in Cuba.

The clinical course, pattern of injury, brain regions involved, cortical and sub-cortical dysfunction,
together with a history of common exposure, all raise the hypothesis of recurrent, low-dose exposure
to neurotoxins. Notably, lesions evident in networks involving the brain stem, basal forebrain and
fornix were highly suggestive of cholinesterase inhibitor intoxication, as the cause of brain injury in
our cohort of Canadian diplomats and families. Biochemical analysis of plasma acetyl- and butyryl-
cholinesterase activity confirmed lower activity in recently-exposed individuals, further supporting
this hypothesis. Toxicological analysis of serum samples using the high-resolution accurate Orbitrap
mass spectrometry confirmed the presence of pyrethroid and organophosphate, and their metabolites
in exposed individuals. While proving the source of exposure and cause of injury is difficult, if not
impossible at this time point, embassy records show a significant increase in fumigation in recent
years with weekly exposure to high dose pesticides in and around many diplomats’ residences. Our
results provide a plausible explanation for acquired brain injury secondary to neurotoxin exposure. A
multidisciplinary approach may offer new mitigation strategies for rapid diagnosis, reduction of
exposure, and potential prevention of future injury.

3
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Dalhousie University | Life Sciences Research Institute | 1348 Summer Street, North Tower | Halifax NS B3H 4R2
www.brainrepair.ca


II Background:
In autumn 2016, some US diplomats posted in Havana reported sensory and auditory stimuli
followed by dizziness, tinnitus and accompanied by cognitive symptoms2. Shortly thereafter,
Canadian government personnel and their families began presenting with similar neurological
symptoms.
Initial testing of Americans (and two Canadians) revealed abnormalities in the subjective visual
vertical test and in both cervical and ocular vestibular evoked myogenic potential metrics. These
results were interpreted as a peripheral vestibular pathology affecting the otolithic organs2.
Approximately 200 days after the suspected exposure, 24 individuals were re-tested at the University
of Pennsylvania (two Canadians were later tested as well). Persistent symptoms included cognitive,
balance, visual and auditory dysfunction; sleep impairment; and headaches. Test findings included
cognitive, vestibular, and oculomotor abnormalities in the majority of individuals1.
In August 2018, Global Affairs Canada (GAC), the Nova Scotia Health Authority (NSHA) and
Dalhousie University agreed to collaborate on this study entitled “Neuroanatomical and
neurofunctional assessment in acquired brain injury” with the goal of using clinical and advanced
research methodologies to investigate possible brain injury in Canadian diplomats and their families.

III Study design:


This study, with both retrospective and prospective components, was approved by the Research
Ethics Board of the NSHA. All participants provided written informed consent. This was a cross-
sectional and longitudinal study to examine behavioral and physiological indices of acquired brain
injury (ABI) in subjects with possible ABI compared to control subjects. Subjects had exposure,
travel and medical histories taken, completed symptom questionnaires, underwent cognitive testing,
gadolinium-enhanced MRI scanning, MEG, and blood testing (referred to as the “screening phase”).
In addition, a “detailed assessment” was performed on symptomatic individuals that included some
or all of the following: a neurological examination, psychiatric interview, visual, orthoptic vestibular,
audiology, and movement assessments. Seven individuals had testing before and after exposure. One
individual was tested twice following exposure to assess longstanding persistent changes.
Subject: 26 adult subjects were referred by Global Affairs Canada (GAC) for evaluation at the Brain
Repair Center, Dalhousie University and NSHA in Halifax, Nova Scotia. Of those, 12 were
considered “non-exposed”, never lived or spent more than 4 weeks in Cuba (usually for vacation or
temporary assignments), 11 were “recently exposed” – as they were tested within one month of
returning from Cuba , and 12 were considered “remotely exposed” - tested 1-19 months after
returning from Cuba (median 14 months). Of the 23 “exposed individuals”, only six individuals
were in Havana during the 2015-2016 period, parallel to the events reported by the Americans. Two
additional subjects who frequently visited Cuba during 2014-2016 for business purposes self-referred
and were also assessed.
Study Procedures (Figure 1):
1. Clinical history: Complete exposure, travel and medical history was taken by the PIs.
2. Anthropometric measures: included waist:hip (W:H) ratio, height, weight (to calculate body
mass index; BMI), blood pressure (BP) and heart rate (HR).
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3. Laboratory testing: serum creatinine, eGFR, and HCG pregnancy test (if applicable) – to
confirm eligibility for contrast-enhanced MRI. In addition, individuals were tested for: fasting
glucose and insulin levels (to determine glucose metabolic status), lipid panel, liver function tests,
complete blood count, thyroid stimulating hormone, and highly sensitive C-reactive protein (hsCRP,
an inflammatory marker for cardiovascular risk). Serum samples were kept frozen for future
assessment of biomarkers. Activity of the enzymes acetylcholinesterase (AChE),
butyrilcholinesterase (BChE) and paraoxonase (PON1) were measured spectrophotometrically
according to published methods3–5.

Figure 1: Study procedures and number of participants

4. Self-rated questionnaires: Individuals answered the following questionnaires (see below for
details): Rivermead Post Concussion Symptom Questionnaire (RPQ), Migraine Disability
Assessment questionnaire (MIDAS), Headache Impact Test (HIT-6), Beck Depression Inventory
(BDI-II), Beck Anxiety Inventory (BAI), Post-Traumatic Stress Disorder Checklist – Civilian (PCL-
5), and Pittsburgh Sleep Quality Index (PSQI). Cognitive functioning was assessed using CANTAB.
4.1. Rivermead post-concussion questionnaire: The Rivermead Post-concussion Symptom
Questionnaire (RPQ)6–8 is a widely used clinical assessment tool designed to measure
symptoms occurring after a traumatic brain injury (TBI). The RPQ is commonly used to
measure severity of symptoms following mild or moderate TBI by presenting 16 symptoms
thought to be common consequences of such an injury. These symptoms, which include
difficulties in cognition/thinking (e.g., memory, concentration), mood or affective
complaints (e.g., depressed mood, irritability, anxiety), and somatic/physiological symptoms
(e.g., dizziness, headache, fatigue, light sensitivity) are often referred to as “post concussive
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syndrome” (PCS)9. For each item the individual is asked to rate the presence of the symptom
over the previous 24 h compared with before the head injury. Symptoms are assessed on a
five-point scale with the response alternatives: never had symptoms (Category 0), have had
symptoms but they have resolved (Category 1), have mild problems with symptoms
(Category 2), have moderate problems with symptoms (Category 3), or have severe
problems with symptoms (Category 4).
4.2. The Migraine Disability Assessment (MIDAS) Questionnaire: The MIDAS questionnaire
captures information on disability in terms of missed days of paid work (or school),
household work (chores), and non-work time (family, social and leisure activities)10. The
MIDAS score was found to be highly reliable in two separate studies conducted in the
United States and the United Kingdom11,12. A MIDAS score < 5 indicates no or little
disability, 6-10 mild disability, 11-20 moderate, and >20 indicates severe disability. In a
study when the MIDAS was validated with physicians’ assessment, the mean score for mild
limitations of activities was 3.6; for moderate limitations, 7; and for severe, 11.3.
4.3. The six-item Headache Impact Test (HIT-6): The HIT-6 was designed to provide a global
measure of adverse headache impact13 and was developed to be used for screening and
monitoring patients with headaches in both clinical practice and clinical research. The HIT-6
items measure the adverse impact of headache on social functioning, role functioning,
vitality, cognitive functioning and psychological distress. The HIT-6 also measures the
severity of headache pain. The six items were selected from 89 items (54 from an existing
adverse headache impact item pool and 35 items recommended by clinicians). The HIT-6
shows good internal consistency and test-retest reliability, construct validity and
responsiveness in general headache patients. Since its initial development and validation, the
HIT-6 has been well received and widely utilized in clinical practice, and applied to clinical
trials for patient screening and treatment monitoring of headaches, including migraine14. The
final HIT-6 score is obtained from simple summation of six items and ranges between 36
and 78, with larger scores reflecting greater impact. Headache impact severity level can be
categorized using score ranges based on the HIT-6 interpretation guide (Bayliss M and
Batenhorst A. The HIT-6TM: a user’s guide. USA: QualityMetric, Inc: Lincoln, RI, 2002.),
The four headache impact severity categories are little or no impact (<=49), some impact
(50–55), substantial impact (56–59), and severe impact (60-78).
4.4. Pittsburgh Sleep Quality Index (PSQI): The PSQI15 is a self-rated questionnaire that assesses
sleep quality and disturbances over a 1-month time interval. Nineteen individual items
generate seven “component” scores: subjective sleep quality, sleep latency, sleep duration,
habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime
dysfunction. The sum of scores for these seven components yields one global score. In a
previous study, a global PSQI score > 5 yielded a diagnostic sensitivity of 89.6% and
specificity of 86.5% (kappa = 0.75, p ⩽ 0.001) in distinguishing good and poor sleepers.
4.5. Beck Depression Inventory - II (BDI-II) and Beck Anxiety Inventory (BAI): Depression is
increasingly being recognized as an important influence on clinical and rehabilitation
outcomes following brain injury. Following mild brain injury, previous literature suggests
that anxiety is present in 3-30% of individuals, and significantly decreases over time in most.
To screen for anxiety and depression, the Beck Anxiety Inventory (BAI) 16 and Beck
Depression Inventory-Second Edition (BDI-II; 17 were administered. Both consist of 21
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items rated on a scale of 0 = not at all, to 3 = severely. Participants endorsed how much they
had been bothered by symptoms of anxiety in the past week and symptoms of depression in
the past 2 weeks. Both the BAI and BDI-II have demonstrated high internal consistency,
test-retest reliability, and convergent and discriminant validity16,18. For the BDI-II,
recommended cut-off scores are 0 to 13 (nil or minimal depressive symptoms), 14 to 19
(mild depressive symptoms), 20 to 28 (moderate depressive symptoms), and 29 to 63 (severe
depressive symptoms). A recent study indicate the BDI-II is useful for identifying symptoms
of depression during TBI recovery19. This study suggested a cut-off score of at least 19 if
one has a mild TBI or at least 35 if one has a moderate or severe TBI. These scores
maximize sensitivity (87%) and specificity (79%). For the BAI - a total score above 22
indicates moderate anxiety and above 35, severe. While the BAI has been validated for
psychiatric populations16, there is little data on its use in patients following brain injury.
4.6. The Posttraumatic Stress Disorder Checklist for the Diagnostic and Statistical Manual-5
(PCL-5): Patients after mild TBI, especially when associated with specific life circumstances
(war veterans, road accidents), often suffer from post-traumatic stress disorder (PTSD) and
post-concussion syndrome (PCS). The PTSD Checklist (PCL) is a widely used DSM-5-
correspondent self-report measure of PTSD symptoms. The PCL was recently revised to
reflect DSM-5 (American Psychiatric Association, 2013) changes to the PTSD criteria. In 2
recent studies20 involving trauma-exposed college students (N = 278 and N=558), PCL-5
scores demonstrated strong reliability and validity. A total score of >33 indicates a likely
diagnosis of PTSD.
5. Cognitive testing (CANTAB): Cognitive functioning was assessed using CANTAB
(www.cantab.com) across the domains of: executive functioning; processing speed; attention;
working memory; and episodic memory.
6. Clinical Neurological assessment: Six subjects underwent full clinical neurological evaluation
which included a thorough neurological history, SCAT-5 and King Devick questionnaires, full
physical examination including mental status, cranial nerves, motor, reflexes, sensory,
coordination and gait assessment.
7. Visual Assessment: A total of 21 subjects (22 assessments) were evaluated for afferent and
efferent visual system defects including infranuclear pathways for cranial nerves 2,3,4 and 6. All
testing was conducted within the Pediatric Ophthalmology and Strabismus unit at the IWK
Health Centre. Testing protocol was approved by the IWK Research Ethics Board. Each
evaluation included a detailed history to determine the presence and nature of any reported visual
symptom.
7.1. General assessment: The afferent visual system was evaluated by visual acuity testing (near
and distance), contrast sensitivity function, and a full evaluation of pupils. Refractive status
was assessed but formal cycloplegic refractions were not conducted. Un-dilated fundus
photographs were obtained and reviewed by an ophthalmologist. Additional tests were done
in the presence of specific signs or symptoms. This included visual electrophysiology,
accommodative function, colour vision, exophthalmometry, intra-ocular pressure (I-Care
device not requiring anesthetic) and reassessment of visual function with appropriate lenses
in place in situations of uncorrected/mis-corrected refractive errors or presbyopia.
7.2. Orthoptic evaluation (emphasis on efferent visual system): This consisted of a detailed
assessment of ocular alignment in all positions of gaze using standardized clinical tests;
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gross assessment of ocular movements with attention to the extent of excursions, and
integrity of eye movement sub-systems (smooth pursuit, saccades, and vergences). Special
attention was directed to convergence ability at near.
7.3. Orthoptic evaluation (emphasis on binocular vision): Testing involved the assessment of
stereo-acuity and fusional amplitudes at distance and near using standardized clinical tests.
7.4. Eye movement recordings: The Eyelink 1000 eye tracking system was used to assess
horizontal saccadic velocities between antagonist extra-ocular muscles and fixation stability
on at central near fixation target.
8. Audiology Assessment: was done according to clinical guidelines at the Nova Scotia Speech and
Hearing Centre. Assessments were performed on 21 (1 no exposure, 10 recent and 10 remote
exposure) subjects and included:
8.1. Pure tone testing. Hearing sensitivity measures were completed in a sound proof booth
according to the American National Standards Institute (ANSI) standards using air and bone
conduction stimuli. Insert transducers were used to deliver pure tones ranging from 250 Hz
to 8000 Hz for both ears. Air conduction masking was employed when the interaural
difference between the two ears at a specific pure tone frequency reached 50 dB. Bone
conduction thresholds were determined at 500, 1000, 2000 and 4000 Hz for each ear and
masking in the opposite ear was used when interaural differences exceeded 10 dB. A
modified Hughson-Westlake approach was used to determine threshold (10 dB down, 5 dB
up bracketed approach) two times out of three for each frequency. Hearing loss severity was
noted to be normal (-10 to 25 dB), mild (26-40 dB), moderate (41 to 55 dB), moderately
severe (56 – 70 dB) severe (71 – 90 dB) or profound (91 + dB). Hearing loss can be either
normal, sensorineural, conductive, or mixed. A few disorders can give pseudo-conductive
hearing losses (dehiscence, large vestibular aqueduct, functional losses) or pseudo-
sensorineural loss (auditory neuropathy) and it is only by combining the results from the
pure tone audiogram, with the information gathered from the middle ear assessment
(tympanograms and acoustic reflexes) along with results from otoacoustic emissions can
these diagnoses be realized. This framework has been called the cross-check principle and is
the hallmark of audiological testing.
8.2. High-frequency (HF) testing included high frequency audiometry (at 9, kHz, 10 kHz,
11.2 kHz, 12.5 kHz, 14 kHz, 16 kHz and 18 kHz). Audiometry was performed with a
Madsen Astera (GN Otometrics, Germany) audiometer with Sennheiser HDA-200 supra-
aural headphones.
8.3. Otoacoustic emissions (OAE’s) is a test of outer hair cell (OHC) integrity of the inner ear.
Research has demonstrated that the OHC’s are capable of movement and the generation of
energy that can be detected as sound in the external auditory canal. Testing requires a
disposable probe to be placed in the ear while the subject is seated quietly in a sound booth.
Stimulation can either be tonal, called distortion products, or click based, or transient evoked
emissions. Results are replicated and compared to normative data. Diagnostic analyses of
OAE findings can be summarized at (a) normal – amplitudes within a designated normal
region, (b), present but abnormal, or (c) not present – absence of OAE activity > 6dB above
the noise floor. It cannot be used to estimate hearing loss and can be absent due to abnormal
middle ear status. The diagnostic applications include separation of cochlear versus
retrocochlear dysfunction, identification of malingering, identification and monitoring of
auditory dysfunction in noise/music exposure, industrial and military hearing screening and
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conservation, diagnosis and management of tinnitus and hyperacusis, and ototoxic
monitoring. For collecting otoacoustic emissions, distortion products were evaluated using a
clinical Distortion Product Otoacoustic Emissions testing (DPOAE) system Scout, Bio-
Logic, Natus. Two primary frequencies, f1 = 65 dB SPL and f2 = 55 dB SPL with a constant
frequency ratio (f2/f1) of 1.2. Responses were measured 750 Hz through 8000 Hz. A
response was considered to be present if it could be measured and replicated with a signal to
noise ratio ≥3 dB. Otoacoustic emissions are either: 1) present and within normal limits, 2)
present and below normal limits, or 3) absent. It is possible for one ear to have all three
conditions.
8.4. Immittance Testing is a combination of impedance and admittance which ascribes to
measure either the opposition at which energy passes through the middle ear system, or
impedance (Z) or its reciprocal, admittance (Y), the ease of flow of acoustic energy through
the middle ear. The measures involve many structures from the external auditory canal
through to the caudal pons of the auditory brainstem for the acoustic reflex. The two
measures used for this study include tympanometry to assess the status of the middle ear and
acoustic reflexes. Tympanometry and acoustic reflex thresholds were performed with a
diagnostic impedance meter Zodiac (Madsen, Natus). Tympanometry was performed using a
226 Hz probe tone of 85 dB SPL to ensure normal middle ear function. Peak compensated
static acoustic admittance (Ymt), tympanometric peak pressure (TPP), and equivalent ear
canal volume (Vea) were recorded. Pump speed was 50 daPa/s with a sweep pressure
starting point of 200 daPa (daPa) and end point of −400 daPa. Tympanometry was obtained
by placing a probe in the subject’s ear canal and measuring change in the tympanic
membrane compliance as a function of pressure change. Various patterns emerge providing
details about the status of the middle ear. When pressure is normal and the movement is
maximal, this yields a typical Type A tympanogram. Type B reveals little or no movement
as the compliance remains relatively unchanged over a large pressure variation. This is
typically found in ears with serous or adhesive otitis media. Type C reveals that the
maximum compliance is shifted to the left of zero, so that is to say, there is negative middle
ear pressure. This finding is common in otherwise healthy ears. The cutoff is -150 daPa.
There are two variants of the normal Type A. Type As and Ad. Type As reveals a normal
pressure with reduced compliance, this is often seen in otosclerosis where the middle ear
ossicles are fused and thus the movement of the tympanic membrane has stiffened. Type Ad
occurs when there is excessive compliance in the middle ear system as can happen with
disarticulation of the middle ear ossicles for example. It is not uncommon for subjects to
have Ad typamograms for reasons such as past history of middle ear infections (most
common), but trauma and past history of tube placement can impact tympanic membrane
compliance as well.
8.5. Acoustic reflexes (AR): The reflex is mediated by the stapedius muscle in the middle ear,
which contracts in response to moderate-to-loud acoustic stimulation, reducing the
immittance of the ossicular chain. The lowest level of acoustic energy needed to produce a
significant change in immittance, generally defined as at least 0.02 mm, is called the acoustic
reflex threshold. The reflex is consensual, when one ear is stimulated, both pathways
respond. Therefore, there are two recording conditions possible: an ipsilateral condition
where the stimulus is presented to the same ear in which the measurement probe is
monitoring immittance, and a contralateral condition where the sound is presented
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contralaterally to the ear in which immittance is being monitored. The reflex is triggered
when an ear is stimulated with a moderately intense signal that reaches the ventral cochlear
nucleus (CN) and is projected via one of two tracts: one leading directly to the facial nerve
nucleus on the same side and another that crosses the midline of the brainstem and connects
with the contralateral superior olivary complex (SOC). The cochlear nucleus also sends a
tract to the ipsilateral SOC which connects to its partner in the contralateral field, as well as
both facial nerve nuclei, both ipsilaterally and contralaterally. The efferent portion of the
pathway from the facial nerve nucleus to the facial nerve runs through the internal auditory
meatus and into the middle ear where it connects with the stapedius muscle. Because of the
complexity of the arc and its involvement with both peripheral and central mechanisms, it
can be impacted by outer, middle, and inner ear pathologies, as well as by pathology of the
auditory nerve and lower brainstem. Reflexes are typically not present for conductive
hearing losses. Ipsilateral and contralateral acoustic reflex thresholds were evaluated at
500 Hz, 1000 Hz, and 2000 Hz separately for each ear. An initial intensity of 80 dB was
used, and the intensity was gradually increased in steps of 5 dB to evoke and confirm the
responses. The acoustic reflex threshold was defined as the lowest stimulus intensity level of
hearibng in dB, at which a reproducible acoustic reflex deflection (representing a minimum
of 0.02 mm change in immittance) from a baseline recording could be detected in two
consecutive trials. For evaluating acoustic reflex thresholds <100 dB HL were considered
normal, whereas thresholds >100 dB HL were considered increased. An absent reflex was
observed when there was no measurable change in immittance (0.02mm) at 105 dB. Testing
was not performed above 105 dB. Reflex decay was measured by raising the intensity 10 dB
about threshold and stimulating the system for 10 seconds between 90 and 105 dB HL.
Abnormal reflex decay was evidenced by the stapedius muscle inability to maintain
maximum contraction (amplitude) for the duration of the stimulus signal. When reflex decay
is present, it is thought to be reflective of eighth cranial nerve dysfunction.
8.6. Behavioral Testing is generally conducted with normal hearing sensitivity. Behavioral
testing can be conducted in subjects with hearing loss, although it requires knowledge about
the type and degree of loss, asymmetries, if the test has been normed for use with hearing
among impairments, among other variables. Tests that have normative data for hearing loss
include dichotic digits, frequency and duration patterns and QuickSin (hearing in noise) test.
Testing can be grouped into low monoaural redundancy speech tests, dichotic speech tests,
binaural interaction speech tests, temporal ordering and resolution. Various combinations of
tests have demonstrated sensitivity and specificity in detecting dysfunction of the central
auditory nervous system and certain tests have a better sensitivity for a particular area of the
central auditory system. For example, if a subject were to have known brainstem
dysfunction, it would be prudent to assess acoustic reflexes, masking level difference and
auditory brainstem response as these tests have demonstrated sensitivity for detecting
brainstem dysfunction. Testing is typically performed at 40-50 dB re: sensation level (SL)
with noted exceptions of QuickSin and Masking level difference performed at 70 dB HL and
competing sentences at 35 dB target ear and 50 dB for competition. Tests utilized; Low
monoaural redundancy speech tests (Compressed speech with reverberation and QuickSin),
Dichotic speech tests (dichotic digits and competing sentences), Binaural interaction speech
tests (masking level difference), and Temporal ordering and resolution (Frequency and
duration patterns, and Gaps in Noise).

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8.7. Auditory Evoked Responses: For auditory evoked testing, surface electrodes were placed on
the head using Ten20 paste and skin-electrode impedance values were maintained below
5 kΩ for all sites. Electroencephalography was monitored and paused when artifacts were
present that might interfere with the recordings. Auditory brainstem response (ABR):
auditory evoked response from a brief acoustic click measuring neural synchrony from
eighth cranial nerve to the upper brainstem (ABR waves I, II, III share the same pathway as
the acoustic reflex pathway). Electrodes are affixed to the subject on the earlobe or mastoid,
the vertex and forehead. Typically, the response generates three waves, I, III and V within
the first 10 msec after stimulation. Generators are thought to be eighth cranial nerve for
waves I and II with cochlear nucleus generating wave III and lateral leminiscus for wave V.
It is thought that for wave III and above, there are multiple generator sites. Comparisons of
absolute latency are made as well as interpeak latencies between waves as well as
comparisons between wave V for both ears. Electrodes were positioned as follows: the active
electrode (Cz) on the vertex, the ground electrode (Fpz) on the forehead, and the reference
ones on the left (M1) and right (M2) mastoids. For ABR testing, the stimulus was click at
80 dB HL, monaural, 11.7 clicks per second, 2000 sweeps, alternating polarity. High pass
filter setup at 100 Hz and a low pass filter at 3000 Hz, and 10 ms window. ABR is a non-
invasive physiological technique used to examine the auditory nerve and
auditory brainstem function. The absolute latencies of waves I, III and V, and interpeak
intervals of I-III, III-V, I-V were analyzed. The measurements were duplicated to ensure
reliability. Auditory middle latency response (AMLR): similar to the ABR using a fast-
auditory click presented via inserts to the ear are used to obtain the MLR occurring between
12 and 70 msec after stimulation. Electrode placement is typically C3 and C4, and at the
earlobes and vertex to derive a Na-Pa response. It can be helpful in detecting both cortical
and subcortical involvement. Inter-subject variations are high and intrasubject measurements
are likely the best method for interpretation. Research has demonstrated that while latency
measures are appropriate, amplitude measures may provide better sensitivity. Generator sites
favour anatomical sites between the inferior colliculus and the auditory cortex, and many
further believe that this response occurs somewhere along the thalamocortical pathway. It is
possible to obtain ear and electrode effects which have been found to yield interesting
findings. An electrode effect is described when the electrode closest to the lesion site yields
the smallest amplitude or the greatest latency when compared with other electrode sites. The
ear effect occurs when regardless of electrode site, stimulation of one ear yields a smaller
amplitude and/or a longer latency than responses obtained by stimulating the other ear. The
ear effect does not necessarily provide information as to lesion site in the same way that the
electrode effect does. The ear effect does not seem to be a strong indicator of which
hemisphere the lesion is located, as it could be contralateral or ipsilateral. (Musiek and
Nagle, 2018). Electrodes were positioned as follows: the ground electrode (Fpz) on the high
forehead, active electrodes were C3 and C4 linked to reference electrodes on the mastoids
(A1 and A2). For MLR testing, the stimulus was click at 70 dB HL, monaural, with 7.1
clicks per second, 1024 sweeps, and alternating polarity. High pass filter setup at 30 Hz and
a low pass filter 1500 Hz, with a 70 ms window. Latencies measures for Na and Pa response
as well as amplitude of Na-Pa complex were obtained. Latencies past 35 msec are
considered abnormal and amplitude differences of 50% or greater are considered abnormal.

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9. Vestibular assessment: was done at the Speech and Hearing Centre of Nova Scotia, according to
clinical guidelines. Assessment was performed in 14 individuals - 8 recently- and 6 remotely-
exposed. Tests included:
9.1. Videonystamography (VNG): Oculomotor (gaze, saccades, tracking, optokinetics),
positional, positioning, and caloric testing were performed using Micromedical Spectrum 9.1
Visual Eyes Four Channel System. Caloric tests were performed using open-loop sequential
bithermal irrigation with water at 30 and 44°C. A caloric weakness of greater than 25% was
regarded as an abnormal decrease on the affected side.
9.2. Cervical and Ocular Vemps: Both oVEMP and cVEMP responses were obtained
using a 500-Hz, 95-dB nHL tone burst, with a 2-cycle rise time, 1- cycle plateau, and a 2-cycle
fall time, gated with a Blackman weighting function using a Biologic Navigator Pro. Through
ER3A (Etymotic Research, Elk Grove Village, Illinois, USA) insert earphones coupled to the
ears with soft compliant foam tips, stimuli were presented at a rate of 5.1 per second. cVEMPs
were recorded from the sternocleidomastoid (SCM) muscles ipsilateral to the stimulated ear in
response to AC 500 Hz TB stimuli. Subjects used a blood pressure manometer to monitor
contraction. Peak-to-peak amplitude between P13 and N23 at 95 dBnHL were measured in µV
and thresholds screened at 70 dBnHL. If present at 70 dBnHL, true thresholds were obtained.
oVEMP recordings were obtained using electrodes placed midline, as close to the lower
margin/belly of the inferior oblique muscle of the lower eyelid as possible, for each eye. While
the active electrodes were placed superiorly, the inverting electrodes were placed 2 to 3 cm
inferiorly. Responses were recorded from the extraocular muscles contralateral to the stimulated
ear in response to AC TB. Patients were placed in a chin rest and asked to look up at a target at
their maximum gaze. The peak-to-peak amplitude in µV of the N1 and P1 waves were
measured. Amplification gain was 100,000.
9.3. Video Head Impulse Test (vHIT): The compensatory eye movement response to a
small, unpredictable, abrupt head impulse was measured by the Micromedical Spectrum 9.1
Visual Eyes System. The same operator delivered every impulse to every subject. The subject
was seated at 1 metre from a television screen with a fixation point at eye level. Head impulses
(peak head velocity 150-300°/s) were performed for lateral canals, Left Anterior Right Posterior
(LARP) /and Right Anterior Left Posterior (RALP) VHIT positions from behind. The VOR gain
reflex was calculated as the ratio of angular eye to head velocity. The vHIT gain of the
vestibulo-ocular reflex was considered normal at ≥0.7 with overt and covert saccades indicating
a peripheral-vestibular deficit.
10. Movement Assessment: Seven individuals from the “remotely” exposed group and three from the
“recently” exposed group were assessed in the Joint Action Research (JAR) Laboratory, School
of Physiotherapy, and the Interprofessional Centre for Attention in Real Environments (iCARE),
School of Occupational Therapy, Dalhousie University. Only preliminary results will be
presented here. Participants completed pre and post iCARE. Measures around the JAR treadmill
protocol. The pre-post iCARE measures included a SR Eyelink 1000 Plus visual flanker test to
gauge efficiency of visual attention for filtering out irrelevant information and suppressing
competing responses; participants then wore SMI ETG 2.0 light-weight mobile eye tracking
glasses participants completed the Metriks Functional Cube waist reach fine dexterity tests in
standing and Trail making A & B in sitting. The Canadian Occupational Performance Measure
(COPM) was used to guide a semi-structured interview to elicit occupations of a self-reported

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‘typical day’. Participants also wore the SMI ETG 2.0 light-weight mobile eye tracking glasses
when completing the JAR 30 minute treadmill test as outlined next. The focus of the assessment
was based on a human movement analysis framework. This included a walking assessment,
muscle strength testing, dynamic balance testing and testing for the electrophysiological
manifestations of muscular fatigue. Testing followed standard methodological details developed
in the lab21–24. Briefly, participants were prepared for surface electromyography (EMG),
consistent with guidelines25 and standard procedures (SENIAM). Skin was lightly shaved and
cleaned with 70% alcohol wipes. Ag/AgCl surface electrodes were placed in a bipolar
configuration over the quadriceps, hamstrings and gastrocnemius. Following, rigid plastic plates
containing four retro-reflective spheres were placed on the trunk, pelvis, lateral femur, lateral
tibia, foot and previously defined anatomical landmarks 26 using velcro straps and secured with
adhesive tape. The retro-reflective spheres were tracked using eight Qualisys OQUS 500 motion
analysis cameras. After a 6-minute warmup on the dual-belt instrumented perturbation treadmill
(R-Mill, MotekForcelink, The Netherlands) 23, participants experienced three repeated series of
eight unexpected medial-lateral, 1 and 3cm perturbations during mid stance at a rate of 0.1m/s on
the right and left limbs. At least 40 unperturbed strides occurred between perturbations. Hip and
knee motions were calculated, and EMG recorded during the perturbations. Three strides
immediately before the perturbation were averaged (T0) and three strides immediately after were
obtained (T1, T2, T3). We also calculated the variability (standard deviation) of the head position
during the walking trials. The walking protocol lasted approximately 30 minutes. Immediately
following, a standard exercise series was completed using a Humac Norm Isokinetic
Dynamometer (Computer Sports Medicine Inc., USA). Knee flexor and extensor strength was
determined, as was the median frequency of the vastus lateralis of both right and left legs. At
least one practice/warm-up contraction was followed by two, three-second maximal isometric
contractions. A 40-second rest period separated each contraction, and standardized verbal
encouragement was given.
11. Magnetic Resonance Imaging (MRI): MRI is currently the structural imaging modality with
the highest resolution for the detection of brain injury. In most cases of mild TBI (or post-concussion
syndrome), however, structural MRI is negative. Several MRI-based methods are being developed to
look at brain fibers, vascular integrity and function as part of clinical research. However, to this day,
none of these methods are available for clinical diagnostic use. All scans were conducted using a 32-
channel head coil on a 3-T GE MR750 MRI scanner.
11.1. Diffusion Tensor Imaging (DTI): DTI data was from 7 unexposed GAC individuals
and 40 normative healthy participants (age 34.00 ± 11.66 SD), 6 from the recently-exposed
group and 8 of the remotely-exposed individuals (age 40.61 ± 9.85 SD).

Diffusion MRI acquisition: Diffusion-weighted MR images were acquired for all participants
(n=65) using, with an Oblique-Axial SS-EPI sequence with magnetic gradients with b-values of
1000 s/mm2 and 60 directions and the following specifications: FOV 22cm, 77 axial slices,
voxel size 2 x 2 x 2 mm3, TR = 8s, and acceleration factor of 2. Image acquisition sequence also
included 7 b=0 interleaved acquisitions to facilitate motion correction. To facilitate image
distortion correction, an additional scan of 7 b=0 images having phase encode blip polarity
reversal was acquired.

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Diffusion MRI Analysis: MRtrix3 software 27 and Fixel-Based Analysis (FBA) 28 were used for
the processing and analysis of the diffusion MRI data. Preprocessing of diffusion images for all
participants (n=65) included denoising, removal of Gibbs ringing artefacts, and correction of
distortions induced by susceptibility off-resonance fields, eddy currents and head motion 29. In
addition, we used bias field correction and global intensity normalization across the normative
healthy participants using group-wise registration to a study-specific FA template. Constrained
spherical deconvolution was used to obtain single-fiber white matter response functions and
averaged across all 40 participants. Obtained response function was used to compute Fiber
Orientation Distribution (FOD) images following the upsampling of diffusion images to voxel
size of 1.3 x 1.3 x 1.3 mm3. A study-specific and unbiased FOD template was generated using
all 40 normative healthy participants. GAC participants were incorporated to the cohort by
correcting for the bias field and performing global intensity normalization using the FA template
generated. FOD images for all participants were then registered to the created template.
Registrations were used to warp subject masks to the template and obtain an intersection
template mask subsequently used for the segmentation of the FOD template into a fixel template
mask. Segmentation involved thresholding the peak amplitude of positive FOD lobes at 0.30 to
constrain the fitting of fixels to white matter fiber populations and mapping obtained fixels to a
fixel mask. Next, subject FOD images were segmented into fixels and values for Fiber Density
(FD), a term used to represent apparent fiber density, were computed per fixel for each subject
following spatial transformation of FOD image to template space. Spatial and orientational
matching between subject and template fixels involved reorientation of all subject fixels using
FOD registration warps and a correspondence process to assign each template fixel to a
matching subject fixel, across all subjects. Using the template fixels and the subject-to-template
Jacobian matrix warps, Fibre Cross-section (FC) was obtained for each fixel across subjects and
then normally distributed by performing a log transformation of the metric. A conjunction of the
two metrics, Fiber Density and Cross-section (FDC), was then computed by multiplying the two
metrics. Whole-brain probabilistic tractography was performed on the FOD template using an
FOD amplitude cutoff of 0.1 to generate 20 million streamlines. The tractogram was then
filtered using Spherical-deconvolution Informed Filtering of Tractograms (SIFT) to 2 million
streamlines to reduce biases from the tractography algorithm. Group statistical comparisons
were performed independently on FD, FC and FDC for every fixel using a General Linear
Model to investigate microstructural differences in white matter tracts between the unexposed
cohort and the exposed cohort. Statistical analysis was conducted using Connectivity-based
Fixel Enhancement (CFE), non-parametric permutation testing using 5000 permutations and
Family-Wise error correction at a p-value of 0.05 and. Age was included in the analysis as a
nuisance covariate. For assessing damage level between the three GAC groups, a mask was
generated for significant fixels and the extracted FD values were compared between unexposed,
recently exposed and remotely exposed GAC participants using t-tests.
11.2 BBB imaging: Participants were intravenously injected with the magnetic contrast-
agent gadoteridol (0.1 mmol/kg, ProHance, Bracco Imaging Canada, Montreal, QC), and its
dynamics in the brain were monitored for a period of 20 minutes using T1-weighted MRI (GE
Discovery MR750, 3T, FOV 24cm, slice thickness 6mm, 192x192 matrix, flip angle 15°, TR/TE
4.1/2.1ms).
Voxel-based BBB assessment was performed as described 30–32. In brief, preprocessing included
image registration and normalization to MNI coordinates using SPM12 (University College
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London, www.fil.ion.ucl.ac.uk/spm). The accumulation rate of the contrast-agent during the
slow enhancement period of the scan (6-20 min) was next calculated for each voxel30–32. To
compensate for physiological (e.g., heart rate, blood flow) and technical (e.g., injection rate)
variabilities between scans, each voxel’s accumulation rate was normalized to that of the
superior sagittal sinus. The normalized contrast-agent accumulation-rates were defined as the
unit-of-measure for BBB permeability, with near-zero/negative values reflecting BBB-protected
tissue and positive values representing tissue with tracer accumulation due to cross BBB
extravasation (Figure 1A). Voxels with abnormally high BBB permeability were identified using
an intensity threshold, previously defined as the 95th percentile of all values in a cohort of
control subjects32. Hence, voxels with normalized-accumulation-rates exceeding 0.02 were
considered as tissue with BBB leakage.
Whole-brain and region-specific BBB assessment: The overall extent of BBB leakage in each
subject’s brain was calculated as the percent of voxels with normalized-accumulation-rates
exceeding 0.02. To quantify region-specific BBB leakage, each scan was segmented into 130
anatomically/functionally significant areas in accordance with the MNI brain atlas (SPM12,
University College London, www.fil.ion.ucl.ac.uk/spm). The extent of BBB leakage within each
region was defined as the percent of region volume with normalized-accumulation-rates
exceeding 0.02.
12. Magnetoencephalography (MEG): Resting state MEG data were collected using an Elekta
Neuromag whole head 306-channel MEG system at the Laboratory for Clinical MEG located at the
IWK Health Center in Halifax, Nova Scotia. Standard operating procedures were utilized for
continuous head position monitoring, head digitization for co-registration of MEG findings to the
anatomical MRI, and the recording of electrooculogram (EOG) and electrocardiogram (ECG) data
for the removal of physiological artifacts. Research participants were scanned in the seated position
in a magnetically shielded room with the active shielding enabled. Two resting-state datasets with a
15 minutes duration each were acquired, one with eyes open and one with eyes closed. MEG data
were sampled continuously during all resting state recordings at 1000 Hz and band-pass filtered at
0.1-330 Hz.
MEG Analysis: Resting state MEG data were analyzed for each subject (non-exposed: N=11; recent
exposure: N=11; remote exposure: N=13). In addition we analyzed age-matched controls (N=61)
including 21 healthy subjects whose data were collected in previous studies and 40 additional healthy
subjects (mean age=42.6 ± 11.15 years), whose data were obtained from the CamCAN repository
(available at http://www.mrc-cbu.cam.ac.uk/datasets/camcan/). MEG data was analyzed offline from
15 minute recordings in 102 channels, at a sampling rate of 1 kHz. The data were preprocessed using
signal space separation 33 to account for active shielding and to remove sources external to the MEG
helmet, down-sampled to 250 Hz and band-pass filtered in the range 1.0-40 Hz. Independent
component analysis (ICA) was used to identify components that are correlated with EOG and ECG
time courses resulting from vertical eye movements and eye blinks, horizontal eye movements and
cardiac signals, and the corresponding components were removed from the data. The remaining ICA
components were visually inspected to determine whether they resulted from artifacts by examining
their time courses, frequency spectrum and topological profiles and additional components were
removed from the data accordingly. The data was then visually inspected in the time domain to
remove time segments containing artifacts. Data in each channel were buffered into 2 sec long
epochs with 1 sec overlap. Spectral analysis by fast furrier transform (FFT) was calculated for each
channel. Bandwidth power (1-3 Hz, 3-8 Hz, 8-12 Hz, 12-20 Hz, 20-50 Hz) was calculated for each
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epoch (2 sec long epochs with 1 sec overlap) in individual channels, averaged across all epochs,
normalized to the total power of the respective channel, and was finally averaged across all channels
for comparison between subjects. Data analysis was performed by in-house Matlab scripts. For each
activity epoch the median-power frequency (MPF) was extracted. Based on our previous studies we
defined periods of paroxysmal slow wave events (PSWEs), as windows of brain activity in which
MPF was lower than 6 Hz for a period longer than 5 sec.
We defined the duration of a non-overlapping event by consecutive seconds in which PSWEs were
occurring in at least 5 channels simultaneously. Occurrence per minute, the accumulative time of
events out of the total recording time, and the mean MPF during events in all channels were
measured.

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IV Results:

1. Demographic and Clinical History: Demographic characteristics are detailed in Table 1. The
three groups were similar in age and sex. Most individuals from the remotely-exposed groups were
staying in Havana significant longer period (median 14 months), compared to the recently-exposed
who generally were tested 5-7 months after posting.

Table 1. Demographic

No exposure Recent exposure Remote exposure


N=12 (%) N = 11 (%) N=14 (%)
Males N (%) 5 (42) 5 (45) 7 (50)

Age, years min, max (mean) 28 – 64 (46.5) 29 – 55 (41.3) 27 – 61 (42.6)

# months in Havana, min-max, (mean) 0 5 – 8 (6.5) 1-48 (27)

Table 2. Common Reported Symptoms*


Domain Symptom No Recent Remote P Value
Exposure Exposure Exposure (Chi squared)

N = 12(%) N = 11 (%) N = 13 (%)


Cognitive Concentration 0 6 (54) 4 (30) 0.0136
Memory 0 3 (27) 7 (53) 0.0110
Dizziness Balance 0 3 (27) 11 (84) <0.0001
Vertigo 1 (8.3) 3 (27) 4 (30) 0.3586
Light 1 (8.3) 3 (27) 4 (30) 0.3586
Headedness
Visual Blurred Vision 1 (8.3) 5 (45) 6 (46) 0.0795
Light Sensitivity 0 2 (18) 5 (38) 0.0521
Auditory/Vestibular Tinnitus 0 2 (18) 9 (69) 0.0005
Sound 0 3 (27) 7 (53) 0.0110
Sensitivity
Vestibular 0 0 6 (46) 0.0017
General Wellbeing Sleep 1 (8.3) 6 (54) 9 (69) 0.0066
Fatigue 0 5 (45) 9 (69) 0.0016
Headaches 0 6 (54) 12 (92) <0.0001
Irritability 0 2 (18) 4 (30) 0.1177
Nausea 3 (25) 3 (27) 5 (38) 0.7359
*
Comments received:

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“Overall symptoms improved, but cognition still bad. Looks at photos but does not recognise people in them.
Trouble driving, can't tell distances. Episode of tingling, top of head, down right side of face for twenty minutes”
“Spontaneous epistaxis” (N=1)
“Difficulty driving at night.” (N=3)

Symptoms reported as part of clinical interview are summarized according to systems in Table 1.
Common symptoms included a general feeling of reduced wellbeing (sleep disturbances, fatigue,
headache and irritability), cognitive impairment (concentration and memory), visual symptoms
(blurred vision and sensitivity to light) and audio-vestibular symptoms (tinnitus, sensitivity to sound
and feeling off balance). While generally symptoms were more often reported by individuals within
the remotely-exposed group, this was not found to be significant, and reported symptoms were
generally similar between the exposed groups.

2. Anthropometric measures: measures were similar to what are usually obtained in the
Canadian population with no differences between the groups (Table 3).
Table 3. Anthropometric measures
No Exposure Recent Exposure Remote Exposure
N = 12 N = 11 N = 13
mean± SEM (%) mean± SEM (%) mean± SEM (%)
BMI 30.15±3.1 30.32± 3.6 (N=9) 28.31±1.3

W:H Ratio 0.88±0.02 0.92±0.02 (N=8) 0.94±0.02

Systolic BP 119.8±5.9 118.7± 6.8 (N=9) 120.7±4.1

Diastolic BP 79.1±2.7 71.1± 4.8 (N=9) 77.5±3.1

Heart Rate 71.3±3.6 73.6± 3.4 (N=9) 74.30±2.4

3. Laboratory testing: measures were similar to what are usually obtained in the Canadian
population with no differences between the groups (Table 4).

Table 4. Routine laboratory tests


No Recent Remote
P Values
Test Units Ref Range Exposure Exposure Exposure
(ANOVA)
N=12 N=11 N=14
Creatine umol/L (49-90) 70.42 76.91 73.07 0.54
mL/min/1.73
eGFR (>60) 88.5 86.64 88.07 0.60
mE2
ALT U/L (0-44) 18.67 18.91 20.23 0.91
AST U/L (5-45) 19.42 20.72 19 0.72
Tri mmol/L (< 1.7) 1.26 1.35 0.82 0.26
Chol mmol/L 5.22 5.08 4.20 0.01
HDL mmol/L 1.46 1.52 1.32 0.33

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LDL 3.09
mmol/L 3.2 2.52 0.06
(N=10)
TSH mlU/L (0.35-4.30) 2.15 2.22 1.82 0.69
FPG mmol/L (3.6-6.0) 5.02 5.12 5.06 0.82
FSI uU/ml 6.57 12.78 9.10 0.36
HOMA-IR 1.48 1.74 2.06 0.18
8.50
hsCRP mg/L 2.90 2.01 0.16
(N=10)
6.58
WBC X10(9)/L (4.50-11.00) 6.03 6.15 0.68
(N=10)
4.99
RBC X10(12)/L (3.80-5.80) 4.62 4.79 0.21
N=10)
147.20
Hgb g/L (120-160) 138.25 141.68 0.33
(N=10)
(0.370- 0.42 0.44 0.43
Hct 0.24
0.470) (N=10)
257.2
PLT X10(9)/L (150-350) 243.58 234.5 0.60
(N=10)

4. Self-rated questionnaires: Of the 8 questionnaires, three were found to be scored more


positively by exposed individuals. Those included the RPQ (for post-concussive syndrome), HIT-6
(for headache severity) and the MIDAS (for migraine) (Table 5).

Table 5. Self-Rated Questionnaires


Recent Remote P value
No Exposure
Domain Questionnaire Exposure Exposure (Chi
N = 12 (%)
N = 11 (%) N = 13 (%) Squared)
Concussion RPQ 2 (16) 5 (45) 9 (69) 0.03
Headaches HIT-6 0 4 (44) N=9 8 (61) 0.0045
Sleep PSQI 7 (58) 8 (72) 8 (66) N=12 0.7542
Migraine MIDAS 0 1 (9) 6 (46) 0.0084
Mental State MMS 0 1 (9) 3 (23) 0.1800
Depression BDI-II 0 1 (9) 3 (23) 0.1687
Anxiety BAI 0 0 (0) 3 (23) 0.0553
PTSD PCL-5 0 1 (9) 1 (7) 0.6005

5. Cognitive assessment: The results of the cognitive assessment (www.cantab.com) were


analyzed across the domains of executive functioning; processing speed; attention; working memory;
and episodic memory, and are summarized in Table 6. Most prominent was the reduction in spatial
working memory within both recently- and remotely-exposed individuals (p=0.0003), while a milder
reduced performance was found in decision making quality.

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Table 6. Cognitive Assessment

TEST Reaction Paired Spatial Multitesting Stockings of Cambridge


Time1 Associates Working Task4 Cambridge5 Gambling
Learning2 Memory3 Task6
Controls 371.9±34.6, 8.9±8.4, 1.3±1.8, 2.7±1.9, 6.0±1.0, 0.98±0.02,
(Mean±SDV, N) 45 45 25 32 34 30
Recent 368.25±20.9, 8.7±11.16, 6.9±7.06, 3.22±2.28, 5.97±0.85, 0.97±0.03,
Exposure 10 10 10 9 8 9
(Mean±SDV, N)
Remote 369.9±29.42, 9.5±7.66, 9.44±7.8, 3.78±3.40, 5.33±0.41, 0.95±0.05,
Exposure 12 12 9 9 9 11
(Mean±SDV, N)
P 0.6 0.9 0.0003 0.5 0.21 0.04

1
Median five choice reaction time; 2 Total Errors (adjusted); 3 Number of errors (across all); 4
Number of incorrect response (total); 5 Mean number of moves; 6 Decision making quality (total
score)

6. Visual assessment:
A total of 22 assessments were performed on 21 subjects over a 4 months period (Table 7).
Non-exposed: Two subjects examined met criteria for this group. Both subjects were asymptomatic
during history taking, however one was found to have abnormal findings on orthoptic testing and eye
recordings. Orthoptic testing revealed an incomitant strabismus evident in up gaze and on side gaze
to either side. This subject appreciated diplopia when specifically asked. The identified strabismus
pattern had no localizing value to support a specific diagnosis. The presence of diplopia suggests this
is acquired i.e. after visual maturity, however no definitive onset can be determined. The general lack
of awareness of diplopia in positions of ocular misalignment suggests this finding is likely not acute.
Eye tracings were also abnormal, however these were difficult to obtain due to his optical correction
which may have resulted in testing artifact. However, a true anomaly cannot be definitely ruled out.
Recent exposure: Ten subjects were included in this group. Five subjects (50%) volunteered visual
symptoms at the time of history taking. Onset was gradual in 3 subjects and acute in 2. Four
symptomatic subjects felt that their symptoms were stable, i.e. not progressive, and 1 reported having
spontaneous resolution. Nine subjects had normal orthoptic findings, of whom 1 had reduced
contrast sensitivity function. This subject was the single individual to report spontaneous resolution
of their previous symptoms. Another subject had abnormal orthoptic findings and abnormal eye
tracings. The orthoptic abnormalities are felt to be attributed to childhood strabismus. Of interest, his
associated symptoms of ‘halos’ around objects cannot easily be attributed to the abnormal orthoptic
abnormalities.
Remote exposure: Nine subjects qualified for this group, and testing including a second visit for one
subject. Seven (of eight) subjects reported visual symptoms at the time of history taking. These were
almost exclusively reported as acute and severe, and were either: (1) acute and stable (N=6); (2)
acute and progressive (N=2); (3) acute and resolved but new symptoms induced following vision
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therapy (1 subject); (4) gradual and stable (1 subject). Two subjects (22%) had abnormal orthoptic
findings: One was also found to have anomalous saccadic velocity tracings, fixation instability and
reduced contrast sensitivity function. This subject had extreme difficulty completing most tasks,
making interpretation challenging. The main feature was episodic accommodative/convergence
spasm mainly manifesting in right gaze that was associated with blur/diplopia. All binocular vision
testing was within normal limits in this subject despite these anomalous findings. The other subject
was determined to have a mild convergence insufficiency that was felt to be exacerbated by her
recent increase in near activities and not using appropriate refractive correction for anisometropia.
Table 7. Ophthalmological Testing

Recent Remote
Exposure Exposure
Test
N = 10(%) N = 10(%)
Orthoptic evaluation 1 (10) 3 (30)
Saccadic velocity 0 N=9 1 (10)
Fixation Stability (90s) 1 (12) N=8 4 (40)
Fundus 0 0
Contrast Sensitivity
Function
1 (10) 1 (10)

7. Audio-vestibular assessment:
Results from the auditory and vestibular assessments are summarized in Tables 8 and 9. Partial
hearing loss was rare and found in 3 of 20 individuals (15%), was asymptomatic and in two was
attributed to a history of sound exposure prior to posting in Cuba. Auditory brain-stem evoked
potential showed long latencies (both absolute and interpeak) in the majority of exposed individuals,
with no significant difference between the two exposed groups. Acoustic reflex was also found to be
positive in 80% of the individuals, in both exposed groups.
The most consistent finding in the vestibular assessment was the presence of low threshold, high-
amplitude cervical and/or ocular vestibular-evoked myogenic potentials (CVEMP and OVEMP,
respectively, see Table 6). This was found in 33-66% of the individuals with no difference between
the recent and remotely-exposed groups
8. Movement assessment:

These data are considered preliminary and due to low sample sizes to date, a qualitative investigation
was pursued. The average age of the group was 43 years and Body Mass Index (BMI) of 25. There
were 6 females and 4 males tested. The average knee strength of these individuals (Knee extensors
2.1Nm/kg; Knee flexors 1.3 Nm/kg and Hip Abductors 1.4 Nm/kg) was found to be between values
previously found for older adults (average age 60) and young adults (average age 25) 22 suggesting
no deficits in knee muscle strength. On average the right leg was slightly stronger than the left leg.
After the walking surface perturbations, knee joint muscle activation 21 and hip/knee biomechanics
typically return to baseline levels and ranges of motion respectively within three strides when older
adults (average age 60) and individuals with knee osteoarthritis (average age 60) are tested. On
average, this return to baseline after three strides was occurring in the current participants with
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exception of two subjects. Table 8 shows the data for all individuals and those who did not return to
baseline are highlighted. We have found the medial 3 cm perturbation to be the most difficult for
individuals to respond to in previous testing. This reduction in dynamic knee and hip motion,
accompanied by greater levels of muscle activation suggest a “stiffening” strategy in the lower
extremity adopted in response to the unexpected perturbation that could not be reduced within three
strides.

Table 8: Results of Audiological Tests

Recent Remote
Test Exposure Exposure P Value
N = 10 (%) N = 10 (%)
ABR Interpeak Latency
(I-III, III-V, I-V) >2.5 8 (80) 6 (60) 0.6
SDV
ABR Interaural Wave V
1 (10) 0 1.0
> 0.3 ms
Absolute latency
6 (60) 5 (50) 1.0
(waves I,III,V) >2.5 SDV
Acoustic Reflexes 8 (80) 8 (80) 1.0

Table 9: Results of Vestibular Tests


Recent Remote
Test Exposure Exposure P Value
N = 9 (%) N = 6 (%)
CVEMP 3 (33.3) 2 (33.3) >0.9999
OVEMP 2 (22.2) 4 (66.6) 0.1357
Caloric & vHIT 1 (11.1) 0 >0.9999
Gaze Nystagmus 0 2 (33.3) 0.1429
Positional 2 (22.2) 0 N=4 >0.9999

Table 10: Knee and hip motion, muscle activation


(Differences from baseline to third stride after perturbation)#

Antagonist
Agonist muscle
Subject Knee motion Hip motion muscle
(Quadriceps)
(Hamstring)
1 -3 -1 0.6 1
2 1 -1 0.4 0
3 0 0 0.2 1
4 -1 -7 1.1 1
5 1 0 0.3 2
6 -10 -12 1.7 6
7 0 1 NaN NaN
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8 1 0 0.6 0
9 2 2 0.5 1
10 0 1 0.2 2
#
Values marked in red would be considered to represent an abnormal response, due to stiffening knee and/or hip joints
for a period after perturbation that was not expected. NaN indicates that EMG was not collected (no consent).

9. Magnetic resonance imaging:


9.1. Clinical MRI: Of the 26 scanned individuals, 4 individuals had non-specific white matter
hyperintensity in T2 and FLAIR sequences. This is a frequent, non-specific finding found in
this age group. In one other individual, a small capillary telangiectasia in the pons was
described, and Arnold Chiari malformation type I was reported in another individual.
Extracranial findings included inflamed air sinuses and mucosal thickening (four
individuals). All the above-mentioned findings are considered incidental with no known
clinical significance.

9.2. Diffusion Tensor Imaging: Figure 2A-B shows streamlines coloured by direction that
correspond to fixels indicating a significant difference between the unexposed and the
exposed groups (p < 0.05). A decrease in FD was observed in the exposed group
predominantly in along the right crus of the fornix, past the hippocampal commissure and
projecting to the hippocampus. Reduced FD was also observed in the splenium of the corpus
callosum. Results obtained from the post-hoc analysis are shown in Figure 2C-D, show an
increase in white matter damage in the affected region in the exposed group. Comparing
between non-exposed recently-exposed GAC participants showed a trend but did not reach
significance (p=0.08). However, when compared to healthy control group, the difference is
significant (p=0.032). The damage observed in the remotely-exposed group is significant
relative to the no-exposed group (p=0.026) and healthy controls (p=0.001).

9.3. Blood-brain barrier integrity: Forty dynamic contrast-enhanced MRIs from 30 individuals
were analyzed, of which 16 non-exposed controls (9 non-GAC controls from a previous
cohort and 7 non-exposed diplomats), 10 within 4 weeks following return from Cuba
(“recent exposure”), 14 individuals were scanned 4 weeks to 48 months after returning from
Cuba. Seven individuals were scanned twice: 6 were scanned prior to their posting in Cuba
and were followed ~6 months later (1-6 days after leaving Havana). Another individual who
was posted for 13 months in the Havana was scanned 2 days after coming back to Canada
and was followed 2 months later (“post exposure”). Data from healthy controls was used to
determine the threshold for abnormal accumulation of the contrast agent (95th percentile)32.
Voxels with above-threshold Gd accumulation were labeled on the T1 scan for each
individual (Figure 3A), and the percentage of brain volume with a leaky BBB (BBB
dysfunction – BBBD) was determined. In addition, following brain registration, percentage
of supra-threshold voxels was measured in 126 brain regions and presented as a Z score
compared to non-exposed controls (Figure 3B).

We first performed statistical comparison on 7 individuals with repeated scans. We measured the
change in the percentage of brain with BBBD and the number of regions with BBBD (p=0.06,

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Wilcoxon). When comparing all brain regions, we found 6 brain regions that statistically showed
an increase in BBBD when scans were taken post-exposure (p<0.05, Figure 3E). Notably, except
for the left pallidum, increased permeability was noted in the right hemisphere, and included the
basal forebrain, anterior insula, posterior orbital, superior frontal and superior occipital gyri
(Figure 3E).

A No exposure > Exposed p<0.05 B Sagittal view posterior fornix


A

C D P=0.026
Fiber density in affected regions

P=0.08
N=48 N=40
N=8 N=6
N=18 N=12

None All Controls None Recent Remote


exposure
Figure 2: Diffusion tensor imaging reveals fiber-specific changes in exposed
individuals: A streamlines coloured by direction that correspond to fixels indicating significant
difference between the unexposed and exposed groups (p < 0.05, age and error-corrected).
A decrease in FD was observed predominantly along the right crus of the fornix, past the
hippocampal commissure and projecting to the hippocampus, as well as in the splenium of
the corpus callosum (B-C). D Results obtained from the post-hoc analysis.

We next compared volume with BBBD within the entire brain and in each region, in recently-
and remotely-exposed groups compared to controls. Generally, while a similar regional pattern of
BBBD was found in both exposed groups, the recently-exposed group showed a more prominent
difference, suggesting the effect of exposure is reversible with time after exposure (Figure 4A).
Overall percentage of brain volume with BBBD was not statistically different between the
groups, while the number of regions with a leaky BBBD (Z score >3) were significantly higher in
both exposed groups (mean±SDV, N: 20.8±25.4, 10; 12.73±6.67, 15; 0.0±0,18 for recently-
exposed, remotely-exposed and controls, respectively). From the 6 regions found leaky in the
post- compared to the pre-exposure group, the right anterior insula and basal forebrain were also

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found leaky in the recently-exposed group, with a difference reaching statistical significance only
for the basal forebrain and the anterior insula in the recently-exposed group.

9. Magnetoencephalography:
MEG was performed in 28 individuals from the present cohort, including 11 non-exposed, 14
recently- and 13 remotely- exposed individuals. In addition, data from 61 age-matched controls was
analyzed, including 11 healthy controls recorded in the same IWK MEG, and 40 age-matched
controls from the Cambridge University collection (see methods). Analysis was performed for a ca.
10 minute recording with eyes closed. Power spectrum analysis revealed an increase in power in the
delta frequency range with decreased alpha (Figure 4A-B). We next searched for events of slow
activity, counting events with a duration with a median frequency is < 6Hz for at least 5 consecutive
seconds (termed paroxysmal slow wave events, PSWEs; Figure XC). The number of PSWEs, the
number of channels in which PSWEs are observed, PSWEs duration and the cumulative relative
duration an individual spent in PSWE.

10. Neuropathological findings in an exposed dog:


An exposed dog belonging to one of the diplomatic families developed changes in behavior during
their time in Havana, specifically aggression and epileptic seizures. Consequently, he was euthanized
in Ottawa on September 2018. Sections from his brain were processed and analyzed by independent
experts in the Koret School of Veterinary Medicine and Weitzman Institute for Science (Rehovot,
Israel). The most pronounced lesions were focal necrosis in the lower midbrain/pons and posterior to
the aquaduct (Figure 5A. circled). Also seen are basophilic linear structures interpreted as damaged
neural processes (arrows in Figure 5B), peri-vascular edema and gliosis. Perivascular hemorrhages
were also seen in multiple areas, including midbrain, the cerebellar and cerebral white matter.
Reactive astrocytes were identified by the presence of visible cytoplasm and positive GFAP staining
(Figure 5E-G). The leakage of serum albumin into cellular structures (mainly astrocytes) and reactive
gliosis further confirms a leaky blood-brain barrier.

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Figure 3: Blood-brain barrier dysfunction following exposure: A. A typical scan from an individual prior
to posting (“pre-exposure”) and 6 months after being in Havana (“post-exposure”). Voxels with a leaky BBB
(>95 percentile of controls) are marked in red. B. Posting in Havana was associated with increase in the
percentage of brain volume with a leaky BBB in most individuals with repeated scans (pre- and post-
exposure). C. Regional-analysis showed brain regions with a leaky BBB > 3 SDVs of controls, mainly in the
right hemisphere. D. The number of leaky regions pre- and post- exposure. E. 6 (out of 126) brain regions
were found statistically different after exposure (p<0.05, corrected for multiple comparisons).

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Figure 4: Blood-brain barrier dysfunction following exposure is region-specific: A. Averaged Z score


in each brain region in recently- and remotely-exposed individuals. Note that while many regions overlap, the
leakage was more pronounced in the recently-exposed group. B. The number of regions with a leaky BBB is
significantly higher in exposed individuals. Among these regions, the right basal forebrain and anterior insula
were found to be most significant.

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Figure 4: Magnetoencephalography confirms paroxysmal slowing of brain


activity: A spectrogram during eyes closed from the same two recently exposed
individuals pre- (left plates) and post- (right plates) exposure. Note the clear dominant
10Hz rhythm prior to posting and intermittent slowing following. B. Group comparison
of spectral analysis (eyes closed) showed a significant reduction in alpha and
increased delta activity in both recent- and remotely-exposed groups. C. Slowing of
activity was composed of paroxysmal slow wave events (PSWEs). The upper trace is
the original MEG recording, and the calculated median frequency below. A PSWE was
defined as the time window in which median frequency was < 6 Hz for > 5 sec. D.
Graphs showing the number of PSWEs/ minute (left), % of channels with PSWEs
(middle) and % of recording time in which individuals spent in PSWEs (right). E. Brain
surface showing spatial distribution of PSWEs.

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Figure 5: Histopathology
of affected dog showing
brain stem lesions and a
leaky blood-brain barrier.
H&E staining showing two
lesions within the lower
midbrain (1) and posterior
to the aquaduct (2). D-E
Perivascular edema was
observed in different
regions including the
midbrain, cerebellum and
cerebral white matter. F-G
immunohistochemical
staining showed
astrogliosis and
accumulation of serum
albumin (white) within
astrocytes (red – GFAP).

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V Summary

The goal of the present study was to document evidence of brain injury from an un-identified cause
in Canadian diplomats and their families who were residing in Cuba, as had been suggested for
American diplomats1. Using a multi-disciplinary, multi-modal and quantitative approach, we report
evidence of brain injury in individuals that were exposed during the years 2015-2017 (and left
Havana during 2018), as well as in individuals who were posted to Havana 6 months prior to testing
(September. – December 2018, “recently exposed”). Our main findings: (1) Reported symptoms
included a general feeling of reduced wellbeing (sleep disturbances, fatigue, headache and
irritability), cognitive impairment (concentration and memory), unspecified visual symptoms
(blurred vision and visual halos) and audio-vestibular symptoms (tinnitus, sensitivity to sound and
feeling off balance). In most individuals, symptoms developed gradually with no clear noticeable
acute precipitating event; (2) Self-reported questionnaires that were statistically different in
exposed individuals included the Rivermead post-concussion symptom questionnaire, which is used
to measure severity of common symptoms reported following traumatic brain injury (e.g difficulties
in cognition, mood or affective complaints and somatic symptoms). Differences related to posting in
Havana were also found on the Migraine Disability Assessment and the Headache Impact
questionnaires, both designed to provide a measure of headache impact. Importantly, no differences
were found between remotely- and recently-exposed individuals, and no significant pathology was
found in other screening questionnaires, including those for anxiety, depression and post-traumatic
stress disorder. Significant disturbances in sleep were found in all groups, regardless of exposure; (3)
Cognitive Testing – showed reduced performance most prominently in spatial working memory,
and milder reduced performance in decision making quality. No differences were found in executive
functioning; processing speed; attention; or episodic memory. No differences were found between
recently- and remotely-exposed groups; (4) Clinical tests revealed no significant pathology in eye
movements or vision testing. Neurological examination was unremarkable. In contrast, the majority
of exposed individuals showed a prolonged latency in auditory brain stem evoked potentials and
positive acoustic reflex. In vestibular assessment, up to 60% of the individuals showed pathological
cervical and/or ocular myogenic potentials with no differences between recently- and remotely-
exposed groups; (5) Movement assessment: preliminary results from ten participants (no control
participants tested yet) showed that all individuals tolerated the testing well, and did not exhibit gross
deviations from what would typically see in the testing. A few individuals responded to the walking
perturbations in an atypical manner based on what has been generally observed previously in healthy
controls. Further testing is required, with greater numbers, to determine significance of these results
and their implications for continued function. (6) Magnetic resonance brain imaging (MRI): while
clinical MRI revealed no consistent or major pathology in exposed individuals, DTI for fiber
tracking showed a significant reduction in apparent fiber density within the right crus of the fornix
and posterior part (splenium) of the corpus callosum. Microvascular pathology and leaky blood-brain
barrier was found more pronounced in both exposed groups, more prominently in recently- compared
with remotely-exposed individuals. Significant vascular pathology was found in individuals scanned
before and after posting in Havana within a 6 months period (recently exposed), and in specific brain
regions, mostly in the right hemisphere. Regional analysis of blood-brain barrier leakage revealed the
right basal forebrain and anterior insula as the regions with significant leaking in exposed individuals
compared with non-exposed controls; (7) Magnetoencephalograophy (MEG): analysis of
recordings of brain activity showed a significant slowing in both exposed groups, more prominently

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in individuals recently-exposed, and over the right cerebral hemisphere. Slowing was due to discrete,
transient slow events that lasted up to 25% of the recording period.

VI Working Medical Diagnosis in Canadians with “Havana Syndrome”


Based on the clinical course, symptoms, clinical tests and imaging data, we propose chronic low
grade cholinesterase inhibitor toxicity as a likely working medical diagnosis underlying then so-
called “Havana syndrome” in Canadians diplomats and their families who were/ are living in Havana
and tested as part of the present study. Here we summarize the clinical, imaging and biochemical
evidence consistent with this diagnosis in light of the medical and scientific literature. While other
causes of brain injury cannot be ruled out, in the absence of a better explanation we propose means
for prevention, screening and follow-up of individuals in the context of exposure to neurotoxins.
Additional toxicological tests are being performed to further support, and perhaps confirm, the
diagnosis and to identify specific agents individuals have been exposed to. Furthermore, such testing
will help develop the means for rapid future assessment.
Cholinesterase (ChE) is one of the key enzymes required for the proper functioning of the nervous
systems in humans, other vertebrates, and insects. ChE breaks down the neurotransmitter
acetylcholine, preventing its action in the junction between nerve and muscles, or between nerve
cells. Certain chemical classes of pesticides, such as organophosphates (OPs) and carbamates work
against undesirable insects by inhibiting the action of ChE. While the effects of cholinesterase
inhibiting products are intended for insect pests, these chemicals can also be poisonous to humans.
Nerve agents (AKA nerve gases) are ChE inhibitors (ChEI) that are too toxic to use as commercial
pesticides - were used as chemical warfare agents and are extremely poisonous. Human exposure to
cholinesterase inhibiting chemicals can result from inhalation, ingestion, or eye or skin contact.
Acute exposure to toxic levels of ChEIs causes a typical clinical presentation within seconds, is
usually easy to recognize, and includes constriction of pupils, profuse salivation, convulsions, and
loss of control of bowel and bladder. If not treated rapidly, there is a high risk of death due to
asphyxia, cardiac arrest, weakness of respiratory muscles and suppression of the brain stem. In
contrast, exposure to low-doses of ChEs is more difficult to diagnose and may not show any
acute symptoms.
Most of the literature on low-level ChE exposure in humans is epidemiological in nature, resulting
from dietary ingestion and occupational exposure34. The major concerns related to such exposure are
delayed effects on the function of the nervous system, and increased prevalence of cognitive,
behavioral and psychomotor dysfunction 35,36. Exposure may have a particularly high impact in
population subgroups such as aged or genetically vulnerable populations (see below). The exact
mechanisms of damage are not known, and additional targets of these toxins (in addition to ChE
inhibition) have been suggested, including hormones, neurotransmitters, neurotrophic factors;
enzymes related to the metabolism of beta amyloid protein as well as neuroinflammation.
The most direct evidence of central effects of low-dose exposure was published by Bowers and
colleagues (1964)37, who analyzed the clinical signs presented by 96 young male volunteers on
active duty in the US Army or the Air Force following their percutaneous exposure to a low dose of
an OP compound likely to be VX. Although the volunteers did not develop overt signs of acute
toxicity, they presented with a syndrome that was broadly referred to as a “state of altered
awareness” and was characterized by difficulty in sustaining attention and slowing of intellectual and
motor processes, in addition to subjective feelings of agitation, anxiety, and confusion 37.
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The 1995 terrorist attack with sarin in the Tokyo subway is the largest documented exposure of a
civilian population to a nerve agent. The attack left 13 people dead and more than 6000 people
complaining of long-term ill health 38,39. Yamasue and colleagues 40reported that 5-7 years after the
attack, victims presented with significant decreases in gray matter volume in the right insular cortex,
the right temporal cortex, and the left hippocampus. In these victims, the volume of the left
subinsular white matter was positively correlated with decreased serum cholinesterase levels
measured after the incident, but not with the occurrence or severity of PTSD 40. Cognitive deficits
were also found 7 years after exposure 41.
About 25–35% of the deployed soldier population from the 1991 Gulf War suffer from
a constellation of inexplicable symptoms referred to as Gulf War Illness (GWI). According to the
Institute of Medicine's report, GWI also known as chronic multi-symptom illness, is defined as the
presence of a spectrum of chronic symptoms experienced for 6 months or longer in at least two of six
categories: development of fatigue, mood and cognitive changes, musculoskeletal
changes, gastrointestinal symptoms, respiratory difficulty, and neurologic abnormalities including
major co-morbidities such as depression and anxiety (Institute of Medicine: Board on the Health of
Select Populations, 2013). The cause for GWI is not known but since symptoms have not been
reported in veterans returning from other military conflicts it is believed that stress is not the major
(or only) factor in the expression of these multi-symptom illnesses. Epidemiological, meteorological
and intelligence data indicated that soldiers were exposed to organophosphate (OP) nerve
agents Sarin and Cyclosarin from fallout released from demolitions of the ammunition dump at
Khamisiyah, Iraq 42,43. After reviewing all the available data, the Research Advisory Committee on
Gulf War Veterans’ Illnesses has strongly implicated exposure to OPs as one of the leading cause for
GWI and neurological dysfunction in veterans 42,44.
The following findings are consistent with low levels of chronic (or repeated) exposure to
ChEIs:
Clinical Course: In contrast with previous reports on American diplomats1,2, most Canadians did not
experience a single triggered event, neither “unexplained sound” or “sensation of pressure”. In most
cases, symptoms developed gradually over the course of their stay in Havana. This course is less
consistent with a single traumatic event injuring the brain (as with “post-concussion syndrome”) and
is more consistent with exposure to a low-level toxin / chemical in which the accumulated exposure
results in symptoms.
Symptoms: As detailed above, major symptoms included headache, visual disturbances
(halos/blurred vision), hearing changes, balance difficulties, and memory and concentration deficits.
While these symptoms overlap with those observed in patients with “post-concussion syndrome”, the
described visual and auditory components are not common after brain injury but have been reported
after exposure to pesticides. Furthermore, the clinical presentation, including cognitive and
psychomotor dysfunction, attention deficits and memory impairment are consistent with reports of
the long-term effects of ChEIs exposure in humans 35,45.
Auditory-Vestibular testing: As reported above, clinical testing showing abnormal brain-stem
evoked potentials and cervical and/or ocular myogenic potentials suggests brain stem malfunction in
exposed individuals. An important cholinergic nucleus is the brain stem pedunculopontine
tegmental nucleus (PPN) (Figure 6). Malfunction of the PPN may have an important role in gait
impairment and balance46–48. Cholinergic innervation is also critically involved in the processing of
auditory input. Acute tinnitus and hearing loss were described in an individual acutely exposed to
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pesticides, indicating cholinergic involvement in central auditory processing 49. Similar to the
findings in the present cohort, chronic exposure to agricultural OPs has been reported to be
associated with auditory dysfunction and delayed brain stem evoked potentials 50,51. Finally, the
vestibular system, through cholinergic fibers, alters hippocampal rhythms and is critically involved
in spatial memory (see below and 52).
Blood-brain barrier integrity: Our research protocol for measuring blood-brain barrier (BBB)
leakage revealed a significant increase in the permeability of brain microvasculature in 6 brain
regions (out of 126), mostly in the right hemisphere, in 7 individuals tested after being in Havana,
compared to their pre-exposure scans. When tested in all exposed compared with non-exposed
individuals, significance differences were found in the right basal forebrain, a region with a key role
in the sleep-awake cycle, arousal, attention and memory and in the right anterior insula, considered
to be associated with sensory processing and body awareness53. The basal forebrain (Figure 6) is a
key cholinergic nucleus, and its involvement strongly supports injury targeting the cholinergic
system. Malfunction of the basal forebrain also explains the reported symptoms, including sleep
disturbances, attention and cognitive decline. Damage to basal forebrain cholinergic neurons has
been reported after acute or chronic intoxication with organophosphates (OPs)54. Another important
cholinergic nucleus is the pedunculopontine tegmental nucleus (PPN) (Figure 6) - which is involved
in mechanisms of arousal and behavioral state control and participates in control of locomotion and
muscle tone. Malfunction of the PPN may have an important role in gait impairment and balance46–
48
.
The reasons for the hemispheric differences and
the dominancy of the right brain in the observed
injury are not known. Interestingly, reduced
volume of the right insular cortex, the right
temporal cortex, and the left hippocampus was
reported by Yamasue et al. (2007) in victims of the
Sarin attack in Tokyo (see below section 2.1 and
40
). This is the first study documenting BBB
pathology in patients before and after acquired
brain injury. Since the methods used are novel,
there are no previous studies on BBB leakage in
humans following intoxication with ChEIs. While
acute poisoning with organophosphate may result Figure 6: the origins and projections of
in seizures and robust BBB opening 55,56, data on major cholinergic neurons
the effects of low-dose exposure is scarce. Several
studies have demonstrated that OPs (e.g. malathion, malaoxon and chlorpyrifos) have a direct effect
on microvasculature, and cause increased permeability in in-vitro models of the BBB, probably due
to reduction in the level of tight junction proteins55,57,58.
Diffusion Tensor Imaging (DTI): Our main DTI finding is a reduction in fiber density within the
right crus of the fornix, past the hippocampal commissure and projecting to the hippocampus. The
predominant injury in the right over left hemisphere is consistent with the microvascular injury
reported above. Furthermore, cholinergic fibers originating in the basal forebrain are projecting via
the fornix to the hippocampus and parahippocampal gyrus, and are critical in spatial memory
processes59, which were found impaired in our cohort (Table 6, and see Figure 2).

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Cortical Slowing: MEG allows the recording of brain neuronal activity, mainly from the cerebral
cortex. A common finding after brain injury is the slowing of brain activity, which can be quantified
using MEG recordings. To this end, we report pathological slowing of brain activity in both exposed
groups, but not in controls. Consistent with the vascular injury discussed above, significant slowing
of activity was found when MEG recordings were compared before and after exposure, further
supporting changes in brain activity due to exposure. The diffuse pattern of slowing observed in our
study (although predominantly right, see Figure 4), support a diffuse change in brain function, as can
be induced by toxins, in contrast to focal injury more characteristic of a traumatic event. Slowing of
brain activity has been reported following exposure to organophosphates, and although not a specific
finding, is consisted with a ChE poisoning 60.
Serum ChE Activity: To further support or rule out the hypothesis of ChE exposure, we measured
the serum activity of the serum enzymes butyryl and acetylcholinesterase, both inhibited by ChE
inhibitors. Indeed, both serum enzymes showed lower serum activity in recently- but not remotely-
exposed individuals (p=0.001), further supporting that individuals posted in Havana are exposed to
toxins from the ChE family.

Figure 7: Cholinesterase biochemical assays in the serum of exposed individuals. Significant


reduction in activity was found in recently-exposed individuals, suggesting exposure to ChE
inhibitors.

Source of exposure: While the source of exposure to toxins of the cholinesterase inhibitor family is
not yet known, both agricultural organophosphates and warfare agents are common and available.
Several nerve agents of the G- [tabun (GA), sarin (GB), chlorosarin (GC) and soman (GD)] and V-
series (VE, VR, VS and VX) have been deployed not only in warfare but also in acts of terrorism 39
and high-profile assassinations61,62. For instance, GB was used in chemical attacks in the Syrian civil
war between 2013 and 2017, while VX was employed to murder the half-brother of the North
Korean leader Kim Jong-un at the Kuala Lumpur Airport in February 2017. A novel class of nerve
agents, the novichoks or the A-series, has recently come into the limelight following the
assassination attempt on the former Russian spy, Sergei Skripal and his daughter Yulia in Salisbury,
UK 63,64. Such events indicate that these nerve agents are available and can be a threat to the
international community. While in most reported incidents, high dose exposure led to acute
poisoning, as stated earlier, low-dose exposure may not be associated with typical acute symptoms
34
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related to ChE inhibition or life-threatening injury 37 and are difficult to rule out. The use of
agricultural pesticides and insecticides is also common in public areas and houses. Fumigation
appears to have increased since 2016, when Cuba “declared war” on the spread of Zika virus (Figure
8 and 65,66). In addition, routine fumigation around and often inside the houses of Canadian diplomats
may have added to extent of exposure (Figure 8).

Why are they not all affected? (Genetic-


environmental interactions and
susceptibility to OPs)
In recent years, there has been an increase
in the understanding of genetic-
environmental interactions that result in
differing susceptibilities to OP poisoning.
Several recent studies have focused on the
ACHE/PON1 locus as a determinant of
inherited susceptibility to environmental
OP exposure3,67. Serum
paraoxonase/arylesterase 1 (PON1) is an
enzyme that in humans is encoded by the
PON1 gene and has paraoxonase activity.
PON1 type Q is the allozyme that most
efficiently hydrolyzes several
organophosphates including sarin, soman,
Figure 8: Fumigation in Havana is common: Indoor fumigation and diazinon. Halley and colleagues
in Havana for the eradication of mosquitos was initiated by the recently demonstrated that ill Gulf War
Cuban government (A, from 65) and the embassy (B). C. number veterans with the GWI neurologic
of signed outdoor fumigation in each residence between 2016- symptom complex were more likely to
2018. Note the sharp increase in frequency during 2017 (mean and have the R allele (heterozygous QR or
range. D. Mean annual number of performed fumigation in each
resident during 2017-2018. Note that most “affected” homozygous R) than to be homozygous Q
(symptomatic) individuals were in the group with high annual for the PON1 gene67. Moreover, low
number of fumigations (Source: Embassy records). “Others” activity of the PON1 type Q distinguished
include individuals who were tested (N=4) and individuals who ill veterans from controls better than the
were not referred by GAC (presumably asymptomatic, N=12). PON1 genotype alone or the activity
levels of the R type. A history of advanced acute toxicity after taking pyridostigmine (a
cholinesterase inhibitor) was also correlated with low PON1 type Q activity. PON1 haplotypes were
also found to be associated with different transcriptional/ translational responses to ChE exposure
(i.e. different serum levels of the enzyme) and abnormal EEG activity in exposed individuals3.

VII Summary
We report the clinical, imaging, and biochemical evidence consistent with the hypothesis of over-
exposure to cholinesterase inhibitors as the cause of brain injury. To further confirm this hypothesis
and potentially study the toxic agent, we initiated collaboration with the National Laboratory of
Toxicology at Dalhousie University, in order to measure traces of possible toxins and their
metabolites in blood and/or urine samples. While other causes cannot be ruled out, our findings point

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to an environmental risk with immediate implications for prevention, screening, and follow-up of
individuals in the context of exposure to neurotoxins. It will be important to review fumigation
procedures, types of agents being used, and environmental levels of exposure. Alternative protective
methods for the prevention of mosquito-borne diseases should be considered.

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