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Abnormal brain response to cholinergic challenge in chronicencephalopathy from the 1991 Gulf War 
Robert W. Haley
a,
⁎
, Jeffrey S. Spence
a,b
, Patrick S. Carmack 
a,b
, Richard F. Gunst 
 b
,William R. Schucany
 b
, Frederick Petty
c,d
, Michael D. Devous Sr.
e
,Frederick J. Bonte
e
, Madhukar H. Trivedi
c
a 
 Epidemiology Division, Departments of Internal Medicine and Clinical Science,University of Texas Southwestern Medical Center, Dallas, TX, USA
 b
 Department of Statistical Science, Southern Methodist University, Dallas, TX, USA
c
 Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX, USA
d
US Department of Veterans Affairs Medical Center, Dallas, TX, USA
e
 Nuclear Medicine Center, Department of Radiology, University of Texas Southwestern Medical, Center, Dallas, TX, USA
Received 11 May 2007; received in revised form 10 April 2008; accepted 6 May 2008
Abstract
Several case definitions of chronic illness in veterans of the 1991 Persian Gulf War have been linked epidemiologicallywith environmental exposure to cholinesterase-inhibiting chemicals, which cause chronic changes in cholinergic receptors inanimal models. Twenty-one chronically ill Gulf War veterans (5 with symptom complex 1, 11 with complex 2, and 5 withcomplex 3) and 17 age-, sex- and education-matched controls, underwent an 99mTc-HMPAO-SPECT brain scan followinginfusion of saline and
N
48 h later a second scan following infusion of physostigmine in saline. From each SPECT image meannormalized regional cerebral blood flow (nrCBF) from 39 small blocks of correlated voxels were extracted with geostatisticalspatial modeling from eight deep gray matter structures in each hemisphere. Baseline nrCBF in symptom complex 2 waslower than controls throughout deep structures. The change in nrCBF after physostigmine (challenge minus baseline) wasnegative in complexes 1 and 3 and controls but positive in complex 2 in some structures. Since effects were opposite indifferent groups, no finding typified the entire patient sample. A hold-out discriminant model of nrCBF from 17 deep brain blocks predicted membership in the clinical groups with sensitivity of 0.95 and specificity of 0.82. Gulf War-associated chronicencephalopathy in a subset of veterans may be due to neuronal dysfunction, including abnormal cholinergic response, in deep brain structures.© 2008 Elsevier Ireland Ltd. All rights reserved.
 Keywords:
Tomography, emission-computed, single-photon; Physostigmine; Brain diseases; Regional blood flow; Persian Gulf syndrome; Veterans
1. Introduction
The nature and causes of the chronic multisymptomillness in veterans of the 1991 Gulf War, officiallydesignated
undiagnosed illness
by the U.S. Department of Veterans Affairs (VA) (Department of Veterans
 Available online at www.sciencedirect.com
Psychiatry Research: Neuroimaging 171 (2009) 207
–
220www.elsevier.com/locate/psychresns
⁎
Corresponding author. Department of Internal Medicine, Uni-versity of Texas Southwestern Medical Center, 5323 Harry HinesBoulevard., Dallas, TX 75390-8874, USA. Tel.: +1 214 648 3075; fax:+1 214 648 7558.
 E-mail address:
Robert.Haley@UTSouthwestern.edu(R.W. Haley).0925-4927/$ - see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.pscychresns.2008.05.004
 
Affairs, 2001), remain controversial. Factor analysis of veterans' typical symptoms have most commonlyidentified three distinct symptom complexes includinga mild cognitive disturbance (complex 1), a moredebilitating neurocognitive state with confusion andvestibular ataxia (complex 2), and a somatic paincondition with continuous joint and muscle aching andcutaneous sensory complaints (complex 3)
—
with allthree groups sharing chronic muscle fatigue, fever,diarrhea, and middle and terminal insomnia, suggestingan overall syndrome with variants (Haley et al., 1997b;Fukuda et al., 1998; Haley, 1999a; Ismail et al., 1999;Haley, 1999b; Haley et al., 2001; Cherry et al., 2001a,b;Bourdette et al., 2001; Kang et al., 2002). Intense debatecontinues over whether these symptom complexesrepresent unique war-related syndromes reflectingidentifiable pathophysiologic mechanisms (Haley,1997; Landrigan et al., 1998; Binns et al., 2004; Grayet al., 2004). The environmental risk factors most commonly linked epidemiologically with the variouscase definitions include organophosphate pesticides, pyridostigmine bromide anti-nerve agent medications,and low-level sarin nerve gas in fallout from Coalition bombing of Iraqi ammunition storage sites
—
all choli-nesterase-inhibiting cholinergic stimulants (Golomb,2008).Numerousanimalexperiments,recentlyreviewed by a Federal advisory committee (Binns et al., 2004),have demonstrated chronic brain changes, includingalteration of cholinergic receptors (Henderson et al.,2001; Henderson et al., 2002), from exposure to thesechemicals either alone or in synergistic combinations.More recently, brain-imaging studies have demonstratedreduced structural volume and fractional anisotropy inmagneticresonanceimagingofbraininJapanesevictimsofthe1995 sarinnerve agent attack intheTokyo subway(Yamasue et al., 2007), and reduced structural volumesin U.S. Gulf War veterans potentially exposed to sarinnerve agent release from ammunition dump demolitionevents immediately after the 1991 Gulf War (Heaton et al., 2007). Both studies found the degree of brainchanges to be correlated with measures of the degree of sarin exposure (e.g., acute blood cholinesterase activitylevels and estimated atmospheric concentrations fromdispersion models). To test the plausibility of anorganophosphate/cholinesterase-inhibitor etiology for chronic encephalopathy in Gulf War veterans, we performed an experiment in which we stimulated athoroughly studied group of chronically ill Gulf War veterans and unaffected control veterans with the short-acting cholinesterase-inhibiting drug physostigmine andassessedtheeffectsoftheresultingcholinergicchallengeon normalized regional cerebral blood flow (nrCBF)measured by single photon emission computed tomo-graphy (SPECT). Our hypothesis was that, if a 1991exposure to cholinesterase-inhibiting chemicals hadaltered cholinergic receptors or their downstreameffecter systems in neurons, the brain activity of affectedveterans would respond differently to a cholinergicchallenge from that of normal veterans with intact cholinergic systems. To address our pre-stated hypoth-esis (Haley et al., 2000b; Meyerhoff et al., 2001; Menonet al., 2004), this article investigates patterns of brainfunction in deep gray matter structures.
2. Methods
2.1. Subjects
The subjects were selected from members of the U.S. Naval construction battalion surveyed in 1995
–
1996 inwhom the three Gulf War symptom complexes weredefined by factor analysis of symptoms (Haley et al.,1997b, 2001). Symptom complex 1 (
“
impaired cogni-tion
”
)isbestcharacterizedbydistractibility,forgetfulness,feeling depressed, and excessive daytime sleepiness(Haleyetal.,2001),andwasepidemiologicallyassociatedwith wearing flea collars containing organophosphate pesticide (Haley and Kurt, 1997). Symptom complex 2(
“
confusion
–
ataxia
”
) involved reduced intellectual func-tioning, confusion, vestibular ataxia/vertigo attacks, andoccasional disorientation (Haley et al., 2001), and wasassociated epidemiologically with exposure to low-levelchemical nerve agent in fallout from bombing of Iraqiammunition depots and adverse effects of pyridostigmineanti-nerve agent prophylaxis (Haley and Kurt, 1997).Symptom complex 3 (
“
central pain
”
) involved joint painin extremities, neck and shoulders, myalgias in arms, and paresthesias/numbness inextremities (Haleyetal.,2001),andwasassociatedwithmorefrequent,heavyapplicationofhighlyconcentratedinsectrepellantandadverseeffectsof pyridostigmine (Haley and Kurt, 1997). Symptomcomplex 2 carried the greatest functional impairment (Haley et al., 1997b, 2002).Eleven subjects with symptom complex 2, five eachwith symptoms complexes 1 and 3, and 17 age-, sex- andeducation-matched control veterans participated in thisSPECT brain scanning experiment between December 1997 and June 1998. One additional symptom complex 2subject was excluded from the analysis because he hadundergone chemotherapy for lymphosarcoma, whichaffected the SPECT results. Prior case-control studiesinvolving these subjects identified differences fromcontrols in brain metabolism measured by magneticresonance spectroscopy (Haley et al., 2000b), central
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 – 
220
 
dopamine turnover (Haley et al., 2000a), autonomicnervous system function (Haley et al., 2004), audioves-tibular function (Haley et al., 1997a; Roland et al., 2000),and genetically determined increased susceptibility to thetoxic effects of cholinesterase-inhibiting organophosphatechemicals (Haley et al., 1999). Symptom complex 2differed more from controls than the other complexes onmost of these tests. All subjects were male members of the same U.S. Naval Reserve construction battalion andsimilar on height, body mass index and educationlevel. The mean ages of the symptom complex 2 and 3groups (53±S.D. 6 yr and 53±7 yr) and the control group(50±7yr)weresimilarbuthigherthanthatofthesymptomcomplex 1 group (39±8 yr). Subjects discontinued allmedications that might affect cerebral blood flow or interact with physostigmine at least three half-lives beforearrivingforthestudyandwerehospitalizedforstudyintheUT Southwestern General Clinical Research Center for 7 days. Controls participated concurrently with ill subjectsthroughout the study.
2.2. Physostigmine challenge and SPECT scanning 
Each subject underwent two SPECT brain scans 2
–
5 days apart. At each brain scanning session subjectsreceived a 60-min intravenous infusion in a semi-darkened room beginning at 1:00 p.m. The intravenousinfusion for the first scan contained only saline as a placebo and that for the second scan contained physostigmine in the same volume of saline. Prior  physostigmine challenge studies obtained cholinergic brain effects with intravenous doses as low as 0.5 mg(Geaney et al., 1990; Hunter et al., 1991), but morecommonly used doses have ranged between 1.0 and2.0 mg (Gustafson et al., 1987; Risch et al., 1981;Janowsky et al., 1986; Furey et al., 2000b). At thesedoses, the maximal physostigmine effect is achieved by40 min into the infusion and persists for at least 40 minmore (Furey et al., 2000a). We used a physostigminedose of 2.0 mg for the initial sets of ill subjects andcontrols but reduced it sequentially to 1.5 mg, 1.25 mgand 1.0 mg over the course of the study to try to reduceunpleasantsideeffects(Fureyetal.,2000a),thoughtonoavail. Ill subjects and well controls were represented ineach dose-level group so that the effects of changingdoses could be evaluated.Attheendoftheinfusion,approximately20mCioftheradiotracerTechnetium-99mhexamethylpropyleneamineoxime (
99m
Tc-HMPAO) had been injected intravenously. Ninety minutes after the
99m
Tc-HMPAO injection,allowing for the radiotracer to be cleared from bloodand facialstructures, a SPECTimage was acquired withaPRISM 3000S three-headed SPECT camera (Picker International, Cleveland) with ultra-high-resolution fan- beam collimators (reconstructed resolution of 6.5 mm), positioned 13
–
13.5 cm from the axis of rotation.Projection data were acquired in a 128×128 matrix in3° increments using a 20% wide centered window (scanduration of 20 min). Image reconstruction was performedin the transverse domain by using back-projection with aramp filter. Voxels in final reconstructed images were1.7×1.7×1.9 mm
3
. The protocol was approved by theinstitutional review board of the University of TexasSouthwestern Medical Center and the U.S. Army HumanSubjects Research Review Board. All subjects gavewritten informed consent before participating.
2.3. Preprocessing of SPECT image data
The study focused primarily on the following eight deep brain structures in each hemisphere consideredlikely
a priori
to be affected in the chronic encephalo- pathy of ill Gulf War veterans: amygdala, caudate head, putamen, globus pallidus, thalamus, hippocampus,midbrain and pons (Haley et al., 2000b; Meyerhoff et al., 2001; Menon et al., 2004).
2.4. Spatial normalization
The spatial normalization program of SPM2 software(Wellcome Department of Imaging Neuroscience,London) was used to transform the SPECT brain imagesof different shapes and sizes to the SPECT template inMNI152 standard space (Chau and McIntosh, 2005).The sets of SPECT images from the baseline and physostigmine sessions were transformed in separate batches with the SPM2 affine-only transformation withthe brain mask turned on to eliminate the high counts inextracerebral tissue. A nuclear medicine specialist reviewed the transformed images for satisfactorynormalization. Two images with unsatisfactory resultswere re-normalized with the SPM2 nonlinear transfor-mation at the standard cutoff of 25 mm using extraheavy regularization constrained to only three iterations(vs. the default of 16) with satisfactory results.
2.5. Count normalization
The intensity of each voxel was scaled to the medianintensity ofall voxelsina largevolumeof white matterinthe centrum semiovale of the same image, yielding aunitless quantity referred to as
normalized regional cerebral blood flow
(
nrCBF 
). This approach was shownto improve signal detection in this image dataset (Spence
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