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Original Contribution |

Cholinergic Autonomic Dysfunction in Veterans With Gulf War Illness Confirmation in a Population-Based Sample FREE

Robert W. Haley, MD; Elizabeth Charuvastra, RN; William E. Shell, MD; David M. Buhner, MD; W. Wesley Marshall, MD; Melanie M. Biggs, PhD; Steve C. Hopkins, BS; Gil I. Wolfe, MD; Steven Vernino, MD, PhD
[+] Author Affiliations

Author Affiliations: Epidemiology Division, Department of Internal Medicine (Drs Haley, Buhner, Marshall, and Biggs), and Neuromuscular Section, Department of Neurology and Neurotherapeutics (Mr Hopkins and Drs Wolfe and Vernino), University of Texas Southwestern Medical Center, Dallas; and Targeted Medical Pharma, Inc, Los Angeles, California (Ms Charuvastra and Dr Shell). Dr Buhner is now with the Department of Mathematical Sciences, University of Texas at Dallas. Dr Marshall is now with Wesmar Solutions, Inc, Frisco, Texas. Dr Wolfe is now with the Department of Neurology, University of Buffalo, The State University of New York.

†Deceased.


JAMA Neurol. 2013;70(2):191-200. doi:10.1001/jamaneurol.2013.596.
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Published online

Background The authors of prior small studies raised the hypothesis that symptoms in veterans of the 1991 Gulf War, such as chronic diarrhea, dizziness, fatigue, and sexual dysfunction, are due to cholinergic autonomic dysfunction.

Objective To perform a confirmatory test of this prestated hypothesis in a larger, representative sample of Gulf War veterans.

Design Nested case-control study.

Setting Clinical and Translational Research Center, University of Texas Southwestern Medical Center, Dallas.

Participants Representative samples of Gulf War veterans meeting a validated case definition of Gulf War illness with 3 variants (called syndromes 1-3) and a control group, all selected randomly from the US Military Health Survey.

Main Outcome Measures Validated domain scales from the Autonomic Symptom Profile questionnaire, the Composite Autonomic Severity Score, and high-frequency heart rate variability from a 24-hour electrocardiogram.

Results The Autonomic Symptom Profile scales were significantly elevated in all 3 syndrome groups (P < .001), primarily due to elevation of the orthostatic intolerance, secretomotor, upper gastrointestinal dysmotility, sleep dysfunction, urinary, and autonomic diarrhea symptom domains. The Composite Autonomic Severity Score was also higher in the 3 syndrome groups (P = .045), especially in syndrome 2, primarily due to a significant reduction in sudomotor function as measured by the Quantitative Sudomotor Axon Reflex Test, most significantly in the foot; the score was intermediate in the ankle and upper leg and was nonsignificant in the arm, indicating a peripheral nerve length–related deficit. The normal increase in high-frequency heart rate variability at night was absent or blunted in all 3 syndrome groups (P < .001).

Conclusion Autonomic symptoms are associated with objective, predominantly cholinergic autonomic deficits in the population of Gulf War veterans.

Figures in this Article

Few medical conditions are as vexing as Gulf War illness to the veterans who experience it, the physicians who are charged with caring for them, and the policy makers who determine the institutional attitudes and level of resources to be directed at the problem. In 1991, the US military deployed 700 000 of the highest-performing members of the all-volunteer army to the Middle East for a 5-week air bombing campaign and a 5-day ground operation involving tank battles and little traditional combat. Yet, an estimated 25% of the force returned with a chronic, often disabling illness involving symptoms of multiple organ systems without obvious physical signs or laboratory abnormalities,1 variously ascribed to fibromyalgia, somatization, deployment stress, chronic fatigue syndrome, adult-onset attention-deficit disorder, or simply multisymptom illness. Evidence from epidemiological and clinical studies suggests a chronic neurotoxic encephalopathy from exposure to cholinesterase-inhibiting chemicals.1,2 A similar chronic illness has been described in pesticide-exposed agricultural workers3 and in survivors of the 1995 subway sarin attack in Tokyo, Japan.4

Among the most troubling reports of the ill veterans are symptoms suggesting autonomic nervous system dysfunction. These include chronic fatigue, pathogen-free diarrhea, delayed gastric emptying and reflux, dizziness, light sensitivity, night sweats, unrefreshing sleep, sexual dysfunction, and an unusually high rate of cholecystitis and cholecystectomy in atypically young male veterans.1 A 2004 study by Haley et al5 measured autonomic function in 21 veterans who fit a factor case definition of 3 syndrome variants and in 17 veteran control subjects (all male) who were matched by age, sex, and education, drawn from an epidemiological survey of a naval reserve unit.6 Spectral analysis of 24-hour Holter electrocardiography demonstrated significant blunting of the normal nocturnal increase in high-frequency heart rate variability (HF HRV), suggesting impaired central control of parasympathetic tone,5 but test results of baroreceptor function, sleep architecture by polysomnography, and sensory and motor nerve conduction were normal. Stein et al7 reported reduced circadian variation in HF HRV among 12 veterans of the Gulf War meeting a modified case definition of multisymptom illness8 recruited from a rheumatology clinic compared with 36 healthy civilian volunteers, but HF HRV reduction was present only in 5 female veterans and not in 6 male veterans with usable HRV measurements. In an evaluation of neuromuscular function in 49 ill British Gulf War veterans and in 26 healthy controls, Sharief et al9 found no differences in quantitative test results of sensory detection thresholds, Valsalva and standing heart rate ratios, and thermoregulatory control of sweating; however, 24-hour HF HRV was not measured. None of these studies provided a thorough description of autonomic symptoms, and none was performed among a population-representative sample of veterans with the full spectrum of Gulf War illness symptoms. The 3 studies5,7,9 are compatible with the possibility of a selective abnormality of central cholinergic parasympathetic control with preserved sympathetic adrenergic and cardiovagal baroreceptor function.

Therefore, we designed a study to test this prestated hypothesis. We evaluated a population-representative sample of Gulf War veterans meeting a validated case definition of Gulf War illness, with a control group and 3 syndrome variants representing the full spectrum of the condition.10

STUDY DESIGN

We studied 97 Gulf War–era veterans, including 66 case veterans with Gulf War illness and 31 control veterans. The participants, randomly selected as a nested case-control study by a 3-stage sample from the US Military Health Survey (Figure 1), were representative of the entire Gulf War–era veteran population (eAppendix). The 66 case veterans met the standardized factor case definition of Gulf War illness, which was previously validated in a clinical sample10 and in a large nationally representative sample.12 Specifically, we studied 21 veterans meeting the factor case definition of Gulf War syndrome 1 (impaired cognition), 24 veterans with syndrome 2 (confusion-ataxia), and 21 veterans with syndrome 3 (central neuropathic pain). The 31 control veterans included 16 who did not meet the factor case definition of Gulf War illness but were deployed to the Kuwaiti theater of operations (deployed controls) and 15 who were in the military during the 1991 Gulf War but were not deployed (nondeployed controls). The demographic characteristics and comorbidities of the final sample are given in Table 1.

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Figure 1. Process for selecting the nested case-control sample of Gulf War veterans suitable for the clinical neuroimaging study of Gulf War illness from the population sample of the US Military Health Survey. Nondeployed control subjects included those who were medically deployable personnel in the US military during the Gulf War but who were not deployed to the Kuwaiti theater of operations and did not meet any of the case definitions for Gulf War illness. In the stage 1 and stage 2 boxes, the differences between the total and the sum across its 5 comparative groups are due to subsyndromic subjects or members of special strata.10 Numbers in the age by sex, race/ethnicity, and officer rank during the war strata in each clinical group are suppressed according to terms of the certificate of confidentiality. The 32 veterans excluded from group misclassification included 31 classified in one of the syndrome groups whose symptoms reported on the survey were not verified by the medical history taken by telephone and 1 classified as a control subject who had omitted symptoms of Gulf War illness on the survey. The 9 excluded for neurological conditions included 5 with a history of traumatic brain injury and 1 each with cerebrovascular disease, Parkinson disease, Guillain-Barré syndrome, and an unspecified chronic disease. The response rate is the American Association for Public Opinion Research response rate 4 and includes in the base the estimated number of eligible cases among those initially selected from the sampling frame to be contacted.11 MR indicates magnetic resonance.

Table Graphic Jump LocationTable 1. Demographic and Comorbidity Measures in Controls and Gulf War Illness Variant Groups
CLINICAL RESEARCH PROTOCOL

All participants were admitted to the University of Texas Southwestern Medical Center's Clinical and Translational Research Center located in Parkland Memorial Hospital, Dallas, where coffee drinking and smoking were allowed to continue. All participants gave written informed consent according to a protocol approved by the institutional review boards of the university. Because all the participants of this nationally representative sample traveled to Dallas for the study, medications could not be discontinued safely until they arrived in the Clinical and Translational Research Center under medical supervision; therefore, medications could be discontinued for only 24 to 48 hours (not necessarily for a full 5 half-lives) before autonomic testing. Whereas full washout is critical for clinical testing of individual participants, potential biasing effects of medication use on group comparisons were tested by multivariable analyses. An experienced clinical psychologist (M.M.B.) interviewed all participants following administration of the Structured Clinical Interview for DSM-IV-TR (SCID)13 and the Clinician-Administered PTSD [posttraumatic stress disorder] Scale (CAPS).14 All investigators who performed or interpreted (E.C., M.M.B., S.C.H., G.I.W., and S.V.) test results were blinded to the participants' case-control status.

Participants initially completed the self-administered Autonomic Symptom Profile (ASP) questionnaire measuring autonomic symptoms, which has been validated in healthy individuals and in patients with autonomic failure.15 Standard weights were applied to construct the Composite Autonomic Symptom Scale (COMPASS) and the subscales of autonomic symptom domains.15 After a 12-hour fast and abstention from alcohol and caffeine, at 7 AM participants underwent the following objective tests of autonomic function in an autonomic laboratory: pupillometry, lacrimation test, the Quantitative Sudomotor Axon Reflex Test,16 heart rate response to deep breathing and Valsalva maneuver, quantitative sensory testing of cooling and heat pain thresholds,17 and blood pressure and heart rate response to head-up tilt with a tilt table (Finapres Monitor; Ohmeda). Details of these tests are provided in the eAppendix

The Composite Autonomic Severity Score (CASS), a standardized semiquantitative score measuring the severity of autonomic dysfunction from 0 (no deficit) to 10 (maximal deficit), was calculated by combining the results of the 3 subsets of the objective autonomic tests and adjusting to standard age and sex. These included sudomotor (range, 0-3), cardiovagal (range, 0-3), and adrenergic (range, 0-4) subsets.16

Twenty-four–hour Holter electrocardiography recordings, performed at home, were digitized at high resolution, and all QRS complexes were reviewed (Pathfinder 710; Reynolds Medical) by a skilled technician who censored aberrant complexes and artifacts. The normal-to-normal R-R intervals in a 5-minute epoch every 15 minutes were analyzed in the frequency domain using a fast Fourier transform algorithm based on the Lomb-Scargle method of spectral analysis18 to produce the standard measures of HF (0.15 to <0.40 Hz), low frequency (0.04 to <0.14 Hz), and very low frequency (0.003 to <0.04 Hz) spectral power, expressed in milliseconds.2 High-frequency HRV is an index mainly of vagal parasympathetic influence on cardiac rhythm and is reproducible over time.19,20

STATISTICAL ANALYSIS

P values are 2-tailed. The reported results were adjusted for age, sex, and race/ethnicity (black vs other). Analyses were rerun to test for confounding by the following covariates: smoking, creatinine clearance, diagnosis of heart disease, glycated hemoglobin level, officer rank during the war, CAPS diagnosis of PTSD, indicators of deconditioning (body mass index and resting pulse rate), and SCID diagnoses of alcohol or other drug abuse or dependence and major depressive disorder, as well as medications the participants were taking, including anticholinergic medications and tricyclic antidepressants.

AUTONOMIC SYMPTOMS

All 3 Gulf War illness variant groups reported significantly more autonomic symptoms, assessed by the ASP, than the control group (Figure 2 and Table 2). The COMPASS scores were significantly elevated for all 3 syndrome groups compared with the controls and were most elevated for syndrome 2 (Figure 2).

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Figure 2. Distribution of values on the Composite Autonomic Symptom Scale (COMPASS) in the control subjects and in the 3 Gulf War illness variant groups. The horizontal bars represent the mean scores. The differences in COMPASS scores across the 4 groups are statistically significant (R2 = 0.59, P < .001).

Table Graphic Jump LocationTable 2. Scores on the Autonomic Symptom Profile Domains Among Control and Gulf War Illness Variant Groups

In the various symptom domains of the ASP (Table 2), the syndrome 2 group had the highest autonomic symptom scores, but the pattern of symptom score elevations was similar among the 3 syndrome groups. The differences between cases and controls explained more variance (R2 ≥ 0.20) in the orthostatic intolerance, secretomotor, upper gastrointestinal dysmotility, sleep dysfunction, and urinary symptom domains and explained less variance (R2 < 0.20) in the pupillomotor, autonomic constipation, vasomotor, male sexual dysfunction, and reflex syncope symptom domains, suggesting deficits related more to cholinergic than adrenergic autonomic systems. Moreover, the group difference on the male sexual dysfunction subscale was mainly due to erectile dysfunction, possibly related to parasympathetic cholinergic control, and not ejaculatory failure, a sympathetic adrenergic function.

OBJECTIVE AUTONOMIC TESTS

On objective autonomic tests, participants with Gulf War illness had significantly more evidence of autonomic deficits than the controls (Table 3). The CASS varied significantly across the clinical groups (P = .045) and was higher in the syndrome 2 group than in the controls (P = .02).

Table Graphic Jump LocationTable 3. Objective Autonomic and Quantitative Sensory Tests in Controls and Gulf War Illness Variant Groupsa

Compared with the controls, all 3 syndrome groups showed significantly reduced distal postganglionic sudomotor function, most significant in the foot, intermediate in the ankle and upper leg, and nonsignificant in the arm, indicating nerve length–related damage to the peripheral autonomic nervous system affecting the distal small cholinergic sudomotor fibers (Table 3). In a multivariable linear model of sudomotor function in the foot controlling for age and race/ethnicity, the case-control difference was significant (P = .02) and did not vary by sex (P = .78 for group × sex interaction). Controlling for the covariates did not alter these findings.

In contrast, no group differences were statistically significant in tests of tear production (Schirmer test), in sympathetic adrenergic function (including the blood pressure responses to Valsalva maneuvers and tilt), or in any of the pupillary measures. These results are summarized in Table 3.

QUANTITATIVE SENSORY TESTS

The syndrome 2 and syndrome 3 groups had increased cooling detection threshold, which was statistically significant only for the syndrome 2 group (Table 3). None of the 3 syndrome groups differed significantly from controls on the heat pain threshold.

CIRCADIAN VARIATION IN PARASYMPATHETIC TONE

From spectral analysis of 24-hour electrocardiogram monitoring, HF HRV increased normally at night in the control group but not in the 3 syndrome groups (Figure 3A and Table 4). In a repeated-measures mixed-effects linear model of log HF HRV, the case-control × day minus night interaction was statistically significant (P < .001), but the 3-way interaction with sex was not (P = .88), indicating that the loss of circadian variation in the 3 syndrome groups compared with the controls was found in both men and women veterans. Controlling for the covariates did not alter these findings.

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Figure 3. Difference in circadian variation of parasympathetic cardiovagal tone between control subjects and 3 Gulf War illness variant groups, measured by spectral analysis of high-frequency heart rate variability (HRV) in 5-minute epochs every hour from 24-hour Holter monitoring electrocardiography. The control group (black circles) is compared with all cases of Gulf War illness (red diamonds) (A) and with syndrome 1 (green triangles), syndrome 2 (red squares), and syndrome 3 (blue stars) (B). The control group showed the expected low cardiovagal tone during the day and a large increase at night. The syndrome 2 group showed depressed tone throughout the 24-hour period, with no evidence of a nocturnal increase. Syndrome 1 and syndrome 3 showed a blunted, delayed increase at night, and syndrome 3 had elevated tone during the day. The statistical test results of the effects in these graphs are given in Table 4.

Table Graphic Jump LocationTable 4. Difference in Circadian Variation of Parasympathetic Cardiovagal Tone Measured by 24-Hour Holter Monitoring Among Gulf War Illness Variant and Control Groupsa

When analyzed by group, all 3 syndrome groups showed significant blunting or loss of the normal nocturnal increase (Figure 3B and Table 4). During the day, HF HRV of the syndrome 1 group did not differ from that of the controls, but the syndrome 2 group had significantly lower HF HRV than the controls, and the syndrome 3 group had significantly higher HF HRV than the controls, particularly during the morning hours (Figure 3B and Table 4).

High-frequency HRV at night was moderately inversely correlated with the CASS index of objective autonomic test results (r = −0.41, P < .001) (Figure 4A). High-frequency HRV during the day was weakly correlated with the CASS (r = −0.22, P = .04) (Figure 4B).

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Figure 4. Correlation of high-frequency heart rate variability (HRV) at night (A) and during the day (B) with the total Composite Autonomic Severity Score (CASS). Normal R-R intervals from a 24-hour Holter monitor electrocardiogram were analyzed by spectral analysis in a 5-minute epoch each hour. For each participant, the hourly measures of the high-frequency spectral component (0.15 to <0.40 Hz) were averaged across the nighttime hours (12 AM to 5 AM) and across the daytime hours (8 AM to 9 PM), and both measures were log transformed. Measurements on the battery of objective autonomic tests were combined to calculate the CASS, on which higher scores indicate greater autonomic impairment.

ASSOCIATION OF AUTONOMIC SYMPTOMS AND OBJECTIVE TEST RESULTS

The COMPASS of all autonomic symptoms was inversely correlated with HF HRV and was directly correlated with the CASS subscales. The correlation was highest with HF HRV during the day and with the CASS sudomotor subscale, and the correlation was lowest with the CASS cardiovagal and adrenergic subscales (Table 5).

Table Graphic Jump LocationTable 5. Partial Spearman Rank Order Correlations of the Total COMPASS Score and Autonomic Symptom Profile Domains With Objective, Laboratory-Based Measures of Autonomic Functiona

The individual symptom domains tended to be correlated with HF HRV or with the CASS sudomotor subscale but not both (Table 5). Specifically, the vasomotor, secretomotor, upper gastrointestinal dysmotility, and pupillomotor symptom domains were most strongly correlated with the CASS sudomotor subscale. The orthostatic intolerance symptom domain was also correlated with the CASS sudomotor subscale, and it was the only symptom domain to be significantly correlated with the CASS adrenergic subscale. In contrast, the upper gastrointestinal dysmotility and sleep dysfunction symptom domains were most strongly associated with HF HRV at night, and the autonomic diarrhea, male sexual dysfunction, and urinary symptom domains were most strongly correlated with HF HRV during the day. Of the 2 components of male sexual dysfunction, erectile dysfunction, a parasympathetic function, was highly correlated with HF HRV during the day, while ejaculatory failure, an adrenergic function, was not. Like ejaculatory failure, reflex syncope was not associated with any of the objective autonomic measures, and these were the only autonomic symptom domains not associated with the 3 syndrome groups (Table 2).

In a nested case-control sample drawn from a national survey in a large representative sample of the Gulf War–era US military population, this study found that a well-validated research case definition of Gulf War illness was strongly associated with standard scales of autonomic symptoms and with objective tests of autonomic dysfunction. Autonomic symptom scores and objective test results were most abnormal compared with the controls in the syndrome 2 group. This reflects the findings of several prior studies in which syndrome 2 consistently was the most disabling10,21 and had the most prominent abnormalities on various objective tests of brain function.2229

The ASP autonomic symptom domains most strongly associated with the case definition tended to be those related predominantly to cholinergic autonomic control, and these symptom domains tended to be most strongly associated with HF HRV measures or with the CASS sudomotor subscale but not with the CASS cardiovagal or adrenergic subscales. On the objective autonomic tests, the 3 syndrome groups differed most from controls on sudomotor testing (Quantitative Sudomotor Axon Reflex Test). The degree of difference on the Quantitative Sudomotor Axon Reflex Test was related to peripheral nerve length, typical of a length-dependent neuropathy of small-caliber, unmyelinated, peripheral nerve fibers. The increased cooling detection thresholds observed in the syndrome 2 group and the syndrome 3 group and described in a previous study30 may also reflect underlying small-fiber impairment.

The autonomic impairment was most clearly demonstrated in the blunting of the normal rise in HF HRV at night. Because peripheral vagal baroreflex function was not significantly impaired, this abnormality of circadian variation in HF HRV suggests dysfunction in the central nervous system control of parasympathetic outflow. The sample size of this study was also sufficient to demonstrate significant, although more subtle, differences in HF HRV among the 3 syndrome groups during the day. Multivariable statistical analyses demonstrated that the objective findings of peripheral sudomotor neuropathy and impaired HF HRV were not explained by smoking, creatinine clearance, psychiatric comorbidity, diagnosis of heart disease, glycated hemoglobin level, officer rank during the war, indicators of deconditioning (body mass index and resting pulse rate), or medications the participants were taking during the period of the study, including anticholinergic medications and tricyclic antidepressants.

The pattern of autonomic symptoms and objective test findings points predominantly to dysfunction of both central and peripheral cholinergic functions, possibly from neurotoxic damage to cholinergic neurons or cholinergic receptors. This proposed explanation is compatible with prior studies27,28 showing that, compared with control subjects, regional cerebral blood flow in veterans with Gulf War illness responds abnormally to cholinergic challenge with physostigmine, suggesting chronic alteration of cholinergic receptors in the brain. Experiments in rodents, undertaken to model the possible chronic effects of sarin in low doses to which Gulf War veterans were exposed in the war, have identified persisting alterations of cholinergic receptors31,32 and of autonomic responses.33

These findings and this explanation are compatible with a prior study9 of neurologic function in ill Gulf War veterans, which found no associations with tests of adrenergic autonomic function and nerve conduction investigations of large-caliber peripheral nerves but generally did not test for circadian variation in HF HRV. Our findings did not confirm the interaction of blunted circadian variation in HF HRV with sex (blunted in women but not in men) reported by Stein et al,7 which may have resulted from their studying a small sample drawn from health care–seeking clients.

This study has several strengths built into the design to avoid weaknesses in past research on Gulf War illness. In contrast to the exploratory nature of a prior study9 of autonomic function in Gulf War veterans, this study was designed as a confirmatory test of a prestated hypothesis raised by previous investigations. The robust sample size and external validity afforded by the nested case-control design drawn from a survey in a large population-representative sample add greater confidence to the findings from prior small studies7,2228 performed in samples from single military units or from clinic volunteers. Particularly important for studying a disease defined by symptoms alone, the case definition of Gulf War illness used in this study is the only one that has been empirically validated by demonstrating a statistically good fit in other Gulf War veteran populations.10,12 Its 3 syndrome variants provide homogeneous clinical groups to maximize statistical power and represent the full spectrum of the illness to determine whether autonomic dysfunction spans the entire spectrum or is limited to part of it. The extensive work by Suarez et al,15 Low,16 and Low et al34 in developing the ASP and the CASS testing systems, used in this study, provided validated measures of autonomic symptom domains and objective autonomic function testing. As in a previous study of autonomic symptoms measured by the ASP,15 the validity of the veterans' symptom reports was supported by correlations of the COMPASS and its domains with the appropriate CASS subscales of objective autonomic test results.

The greatest challenge in our study was the logistical difficulty of selecting and obtaining participation in a lengthy clinical evaluation of non–treatment-seeking veterans with the full spectrum of the Gulf War illness and representative of the population of Gulf War veterans. To accomplish this, the cases and controls sampled from the nationally representative US Military Health Survey were screened by a physician (R.W.H.) who called them by telephone to ensure correct classification on the case definition before participants were enrolled. The medical screening found that 31 of 132 cases (23.5%) and 1 of 53 controls (1.9%) who were selected and contacted were misclassified on the case definition. While some degree of misclassification is present in any epidemiological case definition, minimizing it through advance medical interviews greatly reduced its adverse effect on the statistical power of this study.

The autonomic measures that differed between cases and controls in this study may prove useful in a strategy for clinical diagnosis of Gulf War illness. Of the objective tests used, the one showing the clearest discrimination among all 3 syndrome groups and the control group was the measurement of circadian variation in HF HRV. When tested with the repeated-measures mixed-effects linear model, which appropriately manages variance of the fixed and random effects, the group discrimination is extremely good. However, when HF HRV measurements in multiple epochs are combined to form a single measure of nighttime HF HRV for each participant, the resulting participant-level means display enough residual variance to reduce the usefulness in clinical diagnosis. Additional research should attempt to reformulate the measure of circadian variation in HF HRV to reduce the variance. Measures of the central nervous system mechanisms upstream from the autonomic dysfunction, such as neuroimaging or electroencephalography of brain function,2629 may also be combined with autonomic testing to improve clinical diagnosis.

Perhaps the most important implications of the findings are those bearing on the long-standing debate about the nature of the Gulf War illness. These results confirm dysfunction among Gulf War veterans of both central control of parasympathetic function and peripheral cholinergic autonomic nerves, further implicating underlying damage to the cholinergic components of the central and peripheral nervous systems.

Correspondence: Robert W. Haley, MD, Epidemiology Division, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-8874 (robert.haley@UTSouthwestern.edu).

Accepted for Publication: July 3, 2012.

Published Online: November 26, 2012. doi:10.1001/jamaneurol.2013.596

Author Contributions:Study concept and design: Haley, Wolfe, and Vernino. Acquisition of data: Haley, Charuvastra, Shell, Buhner, Marshall, Biggs, Hopkins, Wolfe, and Vernino. Analysis and interpretation of data: Haley, Shell, Biggs, Wolfe, and Vernino. Drafting of the manuscript: Haley, Shell, Hopkins, and Vernino. Critical revision of the manuscript for important intellectual content: Haley, Charuvastra, Shell, Buhner, Marshall, Biggs, Wolfe, and Vernino. Statistical analysis: Haley and Shell. Obtained funding: Haley. Administrative, technical, and material support: Haley, Charuvastra, Buhner, Marshall, Biggs, Hopkins, and Wolfe. Study supervision: Haley.

Conflict of Interest Disclosures: Dr Haley received an honorarium from Targeted Medical Pharma, Inc, for critical review of a Food and Drug Administration new drug application for a nonpharmaceutical medication to treat fatiguing illness of possible benefit to Gulf War veterans.

Funding/Support: This study was supported by Indefinite Delivery Indefinite Quantity contract VA549-P-0027, awarded and administered by the Department of Veterans Affairs Medical Center, Dallas, Texas; by grant DAMD17-01-1-0741 from the US Army Medical Research and Materiel Command; and by grant UL1RR024982-05, titled North and Central Texas Clinical and Translational Science Initiative, from the National Center for Research Resources, a component of the National Institutes of Health (NIH) and NIH Roadmap for Medical Research.

Role of the Sponsor: The funding agencies had no involvement in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.

Disclaimer: The content does not necessarily reflect the position or the policy of the federal government or the sponsoring agencies, and no official endorsement should be inferred.

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Burger AJ, Charlamb M, Weinrauch LA, D’Elia JA. Short- and long-term reproducibility of heart rate variability in patients with long-standing type I diabetes mellitus.  Am J Cardiol. 1997;80(9):1198-1202
PubMed   |  Link to Article
Haley RW, Maddrey AM, Gershenfeld HK. Severely reduced functional status in veterans fitting a case definition of Gulf War syndrome.  Am J Public Health. 2002;92(1):46-47
PubMed   |  Link to Article
Haley RW, Hom J, Roland PS,  et al.  Evaluation of neurologic function in Gulf War veterans: a blinded case-control study.  JAMA. 1997;277(3):223-230
PubMed   |  Link to Article
Hom J, Haley RW, Kurt TL. Neuropsychological correlates of Gulf War syndrome.  Arch Clin Neuropsychol. 1997;12(6):531-544
PubMed
Roland PS, Haley RW, Yellin W, Owens K, Shoup AG. Vestibular dysfunction in Gulf War syndrome.  Otolaryngol Head Neck Surg. 2000;122(3):319-329
PubMed   |  Link to Article
Haley RW, Marshall WW, McDonald GG, Daugherty MA, Petty F, Fleckenstein JL. Brain abnormalities in Gulf War syndrome: evaluation with 1H MR spectroscopy.  Radiology. 2000;215(3):807-817
PubMed
Tillman GD, Green TA, Ferree TC,  et al.  Impaired response inhibition in ill Gulf War veterans.  J Neurol Sci. 2010;297(1-2):1-5
PubMed   |  Link to Article
Haley RW, Spence JS, Carmack PS,  et al.  Abnormal brain response to cholinergic challenge in chronic encephalopathy from the 1991 Gulf War.  Psychiatry Res. 2009;171(3):207-220
PubMed   |  Link to Article
Li X, Spence JS, Buhner DM,  et al.  Hippocampal dysfunction in Gulf War veterans: investigation with ASL perfusion MR imaging and physostigmine challenge.  Radiology. 2011;261(1):218-225
PubMed   |  Link to Article
Tillman GD, Calley CS, Green TA,  et al.  Event-related potential patterns associated with hyperarousal in Gulf War illness syndrome groups.  Neurotoxicology. 2012;33(5):1096-1105
PubMed  |  Link to Article   |  Link to Article
Jamal GA, Hansen S, Apartopoulos F, Peden A. The “Gulf War syndrome”: is there evidence of dysfunction in the nervous system?  J Neurol Neurosurg Psychiatry. 1996;60(4):449-451
PubMed   |  Link to Article
Jones KH, Dechkovskaia AM, Herrick EA, Abdel-Rahman AA, Khan WA, Abou-Donia MB. Subchronic effects following a single sarin exposure on blood-brain and blood-testes barrier permeability, acetylcholinesterase, and acetylcholine receptors in the central nervous system of rat: a dose-response study.  J Toxicol Environ Health A. 2000;61(8):695-707
PubMed   |  Link to Article
Henderson RF, Barr EB, Blackwell WB,  et al.  Response of rats to low levels of sarin.  Toxicol Appl Pharmacol. 2002;184(2):67-76
PubMed   |  Link to Article
Morris M, Key MP, Farah V. Sarin produces delayed cardiac and central autonomic changes.  Exp Neurol. 2007;203(1):110-115
PubMed   |  Link to Article
Low PA, Denq JC, Opfer-Gehrking TL, Dyck PJ, O’Brien PC, Slezak JM. Effect of age and gender on sudomotor and cardiovagal function and blood pressure response to tilt in normal subjects.  Muscle Nerve. 1997;20(12):1561-1568
PubMed   |  Link to Article

Figures

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Figure 1. Process for selecting the nested case-control sample of Gulf War veterans suitable for the clinical neuroimaging study of Gulf War illness from the population sample of the US Military Health Survey. Nondeployed control subjects included those who were medically deployable personnel in the US military during the Gulf War but who were not deployed to the Kuwaiti theater of operations and did not meet any of the case definitions for Gulf War illness. In the stage 1 and stage 2 boxes, the differences between the total and the sum across its 5 comparative groups are due to subsyndromic subjects or members of special strata.10 Numbers in the age by sex, race/ethnicity, and officer rank during the war strata in each clinical group are suppressed according to terms of the certificate of confidentiality. The 32 veterans excluded from group misclassification included 31 classified in one of the syndrome groups whose symptoms reported on the survey were not verified by the medical history taken by telephone and 1 classified as a control subject who had omitted symptoms of Gulf War illness on the survey. The 9 excluded for neurological conditions included 5 with a history of traumatic brain injury and 1 each with cerebrovascular disease, Parkinson disease, Guillain-Barré syndrome, and an unspecified chronic disease. The response rate is the American Association for Public Opinion Research response rate 4 and includes in the base the estimated number of eligible cases among those initially selected from the sampling frame to be contacted.11 MR indicates magnetic resonance.

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Figure 2. Distribution of values on the Composite Autonomic Symptom Scale (COMPASS) in the control subjects and in the 3 Gulf War illness variant groups. The horizontal bars represent the mean scores. The differences in COMPASS scores across the 4 groups are statistically significant (R2 = 0.59, P < .001).

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Figure 3. Difference in circadian variation of parasympathetic cardiovagal tone between control subjects and 3 Gulf War illness variant groups, measured by spectral analysis of high-frequency heart rate variability (HRV) in 5-minute epochs every hour from 24-hour Holter monitoring electrocardiography. The control group (black circles) is compared with all cases of Gulf War illness (red diamonds) (A) and with syndrome 1 (green triangles), syndrome 2 (red squares), and syndrome 3 (blue stars) (B). The control group showed the expected low cardiovagal tone during the day and a large increase at night. The syndrome 2 group showed depressed tone throughout the 24-hour period, with no evidence of a nocturnal increase. Syndrome 1 and syndrome 3 showed a blunted, delayed increase at night, and syndrome 3 had elevated tone during the day. The statistical test results of the effects in these graphs are given in Table 4.

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Figure 4. Correlation of high-frequency heart rate variability (HRV) at night (A) and during the day (B) with the total Composite Autonomic Severity Score (CASS). Normal R-R intervals from a 24-hour Holter monitor electrocardiogram were analyzed by spectral analysis in a 5-minute epoch each hour. For each participant, the hourly measures of the high-frequency spectral component (0.15 to <0.40 Hz) were averaged across the nighttime hours (12 AM to 5 AM) and across the daytime hours (8 AM to 9 PM), and both measures were log transformed. Measurements on the battery of objective autonomic tests were combined to calculate the CASS, on which higher scores indicate greater autonomic impairment.

Tables

Table Graphic Jump LocationTable 1. Demographic and Comorbidity Measures in Controls and Gulf War Illness Variant Groups
Table Graphic Jump LocationTable 2. Scores on the Autonomic Symptom Profile Domains Among Control and Gulf War Illness Variant Groups
Table Graphic Jump LocationTable 3. Objective Autonomic and Quantitative Sensory Tests in Controls and Gulf War Illness Variant Groupsa
Table Graphic Jump LocationTable 4. Difference in Circadian Variation of Parasympathetic Cardiovagal Tone Measured by 24-Hour Holter Monitoring Among Gulf War Illness Variant and Control Groupsa
Table Graphic Jump LocationTable 5. Partial Spearman Rank Order Correlations of the Total COMPASS Score and Autonomic Symptom Profile Domains With Objective, Laboratory-Based Measures of Autonomic Functiona

References

 US Department of Veterans Affairs. Research Advisory Committee on Gulf War Veterans' Illnesses. http://www1.va.gov/rac-gwvi/. Accessed October 1, 2012
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American Association for Public Opinion Research.  Standard Definitions: Final Dispositions of Case Codes and Outcome Rates for Surveys. 5th ed. Lenexa, KS: American Association for Public Opinion Research; 2008
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Burger AJ, Charlamb M, Weinrauch LA, D’Elia JA. Short- and long-term reproducibility of heart rate variability in patients with long-standing type I diabetes mellitus.  Am J Cardiol. 1997;80(9):1198-1202
PubMed   |  Link to Article
Haley RW, Maddrey AM, Gershenfeld HK. Severely reduced functional status in veterans fitting a case definition of Gulf War syndrome.  Am J Public Health. 2002;92(1):46-47
PubMed   |  Link to Article
Haley RW, Hom J, Roland PS,  et al.  Evaluation of neurologic function in Gulf War veterans: a blinded case-control study.  JAMA. 1997;277(3):223-230
PubMed   |  Link to Article
Hom J, Haley RW, Kurt TL. Neuropsychological correlates of Gulf War syndrome.  Arch Clin Neuropsychol. 1997;12(6):531-544
PubMed
Roland PS, Haley RW, Yellin W, Owens K, Shoup AG. Vestibular dysfunction in Gulf War syndrome.  Otolaryngol Head Neck Surg. 2000;122(3):319-329
PubMed   |  Link to Article
Haley RW, Marshall WW, McDonald GG, Daugherty MA, Petty F, Fleckenstein JL. Brain abnormalities in Gulf War syndrome: evaluation with 1H MR spectroscopy.  Radiology. 2000;215(3):807-817
PubMed
Tillman GD, Green TA, Ferree TC,  et al.  Impaired response inhibition in ill Gulf War veterans.  J Neurol Sci. 2010;297(1-2):1-5
PubMed   |  Link to Article
Haley RW, Spence JS, Carmack PS,  et al.  Abnormal brain response to cholinergic challenge in chronic encephalopathy from the 1991 Gulf War.  Psychiatry Res. 2009;171(3):207-220
PubMed   |  Link to Article
Li X, Spence JS, Buhner DM,  et al.  Hippocampal dysfunction in Gulf War veterans: investigation with ASL perfusion MR imaging and physostigmine challenge.  Radiology. 2011;261(1):218-225
PubMed   |  Link to Article
Tillman GD, Calley CS, Green TA,  et al.  Event-related potential patterns associated with hyperarousal in Gulf War illness syndrome groups.  Neurotoxicology. 2012;33(5):1096-1105
PubMed  |  Link to Article   |  Link to Article
Jamal GA, Hansen S, Apartopoulos F, Peden A. The “Gulf War syndrome”: is there evidence of dysfunction in the nervous system?  J Neurol Neurosurg Psychiatry. 1996;60(4):449-451
PubMed   |  Link to Article
Jones KH, Dechkovskaia AM, Herrick EA, Abdel-Rahman AA, Khan WA, Abou-Donia MB. Subchronic effects following a single sarin exposure on blood-brain and blood-testes barrier permeability, acetylcholinesterase, and acetylcholine receptors in the central nervous system of rat: a dose-response study.  J Toxicol Environ Health A. 2000;61(8):695-707
PubMed   |  Link to Article
Henderson RF, Barr EB, Blackwell WB,  et al.  Response of rats to low levels of sarin.  Toxicol Appl Pharmacol. 2002;184(2):67-76
PubMed   |  Link to Article
Morris M, Key MP, Farah V. Sarin produces delayed cardiac and central autonomic changes.  Exp Neurol. 2007;203(1):110-115
PubMed   |  Link to Article
Low PA, Denq JC, Opfer-Gehrking TL, Dyck PJ, O’Brien PC, Slezak JM. Effect of age and gender on sudomotor and cardiovagal function and blood pressure response to tilt in normal subjects.  Muscle Nerve. 1997;20(12):1561-1568
PubMed   |  Link to Article

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Haley RW, Charuvastra E, Shell WE, et al. Cholinergic autonomic dysfunction among veterans with Gulf War syndrome: confirmation in a population-based sample. JAMA Neurol.. Published online November 21, 2012. doi:10.1001/jamaneurol.2013.596.

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