Chronic cigarette smoking modulates injury and short-term recovery of the medial temporal lobe in alcoholics

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aging 162 (2008) 133–145www.elsevier.com/locate/psychresns

Psychiatry Research: Neuroim

Chronic cigarette smoking modulates injury and short-termrecovery of the medial temporal lobe in alcoholics

Stefan Gazdzinskia,⁎, Timothy C. Durazzoa, Ping-Hong Yeha, Dawn Hardina,Peter Banysb,c, Dieter J. Meyerhoff a,b

aMagnetic Resonance Unit, San Francisco Veterans Administration Medical Center, San Francisco, CA, United StatesbDepartment of Radiology, University of California, San Francisco, CA, United StatescDepartment of Psychiatry, University of California, San Francisco, CA, United States

Received 3 November 2006; received in revised form 16 February 2007; accepted 8 April 2007

Abstract

Memory function is largely mediated by the medial temporal lobe (MTL), and its compromise has been observed in alcoholdependence and chronic cigarette smoking. The effects of heavy alcohol consumption and chronic smoking on hippocampalvolumes and MTL metabolites and their recovery during abstinence from alcohol have not been assessed. Male alcoholics intreatment (ALC) [13 smokers (sALC) and 11 non-smokers (nsALC)] underwent quantitative magnetic resonance imaging andshort-echo proton magnetic resonance spectroscopic imaging at 1 week and 1 month of sobriety. Outcome measures werecompared with 14 age-matched, non-smoking light-drinkers and were related to visuospatial learning and memory. Over 1 monthof abstinence, N-acetyl-aspartate, a neuronal marker, and membrane-associated choline-containing metabolites normalized in theMTL of nsALC subjects, but remained low in the MTL of sALC subjects. Metabolite concentration changes in both groups wereassociated with improvements in visuospatial memory. Hippocampal volumes increased in both groups during abstinence, butincreasing volumes correlated with visuospatial memory improvements only in nsALC subjects. In summary, chronic cigarettesmoking in alcohol-dependent men appears to have adverse effects on MTL metabolite recovery during short-term sobriety. Thesedata may also have implications for other conditions with established MTL involvement and significant smoking co-morbidity,such as schizophrenia-spectrum and mood disorders.© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Hippocampus; Magnetic resonance imaging; Magnetic resonance spectroscopy; Abstinence from alcohol; Learning and memory;Recovery

1. Introduction

The hippocampus and themedial temporal lobe (MTL)play an important role in encoding and consolidation of

⁎ Corresponding author. San Francisco Veterans AdministrationMedical Center, 4150 Clement Street (114M), San Francisco, CA94121, United States. Tel.: +1 415 221 4810x2553; fax: +1 415 668 2864.

E-mail address: [email protected] (S. Gazdzinski).

0925-4927/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights resedoi:10.1016/j.pscychresns.2007.04.003

new declarative memories as well as encoding and re-trieval of the spatial and temporal context of personal lifeevents (Jarrard, 1995).Alcohol use disordersmay result inneurocognitive deficits consistent with hippocampal and/or MTL dysfunction (Sullivan et al., 2000; Crews et al.,2005). Previous magnetic resonance studies in 1-month-abstinent alcohol-dependent individuals demonstratedvolume reductions in the bilateral hippocampi (Beresfordet al., 2006; Bleich et al., 2003; Sullivan et al., 1995;

rved.

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Agartz et al., 1999; Laakso et al., 2000), with somesuggesting differential effects for sex and/or hemisphere(Agartz et al., 1999; Laakso et al., 2000). Small neuro-pathological studies found unchanged numbers of hippo-campal neurons in alcoholics (Korbo, 1999; Harding et al.,1997), but profound loss in glial cell populations (Korbo,1999) and hippocampal white matter (WM) volume(Harding et al., 1997). MRI volumetric studies, however,cannot distinguish between injury to neuronal or glialcells, and they are not sensitive to tissue density changesreported in recovering alcoholics (Trabert et al., 1995).Proton magnetic resonance spectroscopy (1H MRS) maydistinguish between neuronal and glial injury and may besensitive to changes in brain tissue density (Martin et al.,1995). We used 1H-MRS at short echo times to measurethe following four major metabolites (Ross and Bluml,2001): (1) N-acetyl-aspartate (NAA), an accepted markerof neuronal viability, is observed only in mature neuronsand their processes; decreased NAA may reflect neuronalloss, atrophied dendrites and axons, and/or derangementsof neurometabolism. (2) Choline-containing compounds(Cho) are believed to be involved in cell membranebreakdown and synthesis. (3) Creatine-containing meta-bolites (Cr) consist of creatine and phosphocreatine, whichare involved in cell bioenergetics. (4) Myo-inositol (m-Ino) is a putative marker of astrocytes and may alsofunction as an osmolyte (Schweinsburg et al., 2000).MRSstudies in recently detoxified alcoholics generally reportedlower NAA and Cho in the cerebellum and frontal lobethat tended to increase with sustained abstinence fromalcohol (Durazzo et al., 2004, 2006; Ende et al., 2005;Jagannathan et al., 1996; Parks et al., 2002; Schweinsburget al., 2001; Seitz et al., 1999;Bendszus et al., 2001).Myo-inositol was elevated in multiple brain regions of 1-monthabstinent alcoholics (Schweinsburg et al., 2000). To date,however, there are no published MRS studies on MTLmetabolites in chronic alcoholics.

Chronic cigarette smoking is a common comorbidcondition in alcohol dependence (Romberger and Grant,2004; Hurt et al., 1994; Pomerleau et al., 1997), illicitsubstance abuse, and schizophrenia-spectrum and mooddisorders (Patkar et al., 2006; Dani and Harris, 2005;Esterberg and Compton, 2005; Fergusson et al., 2003).While nicotine may acutely facilitate learning andmemory (Levin and Simon, 1998; Newhouse et al.,2004; Sacco et al., 2004), a growing body of evidencesuggests chronic cigarette smoking in non-alcoholicindividuals adversely affects multiple domains of neuro-cognition (e.g., Deary et al., 2003; Razani et al., 2004),including learning and memory (Heffernan et al., 2005;Hill et al., 2003; Richards et al., 2003; Schinka et al.,2003). Chronic cigarette smoking, in addition to alcohol,

results in additional oxidative stress to brain cells(Moriarty et al., 2003), and cigarette smoke containsmany toxic compounds (Fowles et al., 2000) that maydirectly or indirectly compromise central nervous systemtissue (Durazzo et al., 2006), possibly leading tovolumetric and/or metabolic abnormalities. Cigarettesmoking in the general population was associated withincreased late-life brain atrophy (e.g., Akiyama et al.,1997), regionally specific gray matter reductions in adults(Brody et al., 2004), reduced MTL NAA (Gallinat et al.,2007), and global cerebral blood flow abnormalities (e.g.,Rourke et al., 1997), possibly associated with compro-mised brainmetabolic activity. Along this line, our studiesin alcohol-dependent individuals demonstrated detrimen-tal effects of chronic cigarette smoking on lobar graymatter (GM) volumes and GM perfusion (Gazdzinskiet al., 2005, 2006), as well as on NAA and Cho concen-trations in the frontal lobes and subcortical structures(Durazzo et al., 2004). Additionally, smoking alcoholicsdemonstrated generally lower recovery of regional NAAand Cho levels compared with non-smokers over 1 monthof abstinence from alcohol (Durazzo et al., 2006).

In this study, we employed MRI and 1H magneticresonance spectroscopic imaging (1H MRSI) to measurehippocampal volumes and mean MTL metabolite con-centrations in a group of alcohol-dependent individuals(ALC) at 1 week and 1 month of abstinence from alcohol.The alcoholics were retrospectively classified into smo-kers (sALC) and non-smokers (nsALC) and comparedwith an age-matched group of non-smoking, light drinkingcontrols (nsLD). We hypothesized that (1) at 1 week ofabstinence, sALC subjects would have smaller hippocam-pal volumes and lower MTL NAA and Cho concentra-tions than both nsLD and nsALC, whereas nsALCsubjects would have smaller hippocampal volumes andlower MTL NAA and Cho concentrations than nsLDsubjects; (2) sALC subjects would show less volumetricand metabolite recovery over 1 month of abstinence thannsALC subjects; (3) at 1 month of abstinence, both sALCand nsALC subjects would demonstrate smaller hippo-campal volumes and lower MTL concentrations of NAAand Cho than nsLD subjects; and (4) hippocampalvolumes and MTL metabolite concentrations and theirchanges with abstinence would be related to measures ofdrinking and smoking severity as well as neurocognition.

2. Methods

2.1. Participants

Twenty-four male alcohol-dependent individuals be-tween the ages of 28 and 66, recruited from the San

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Francisco VA Medical Center Substance Abuse DayHospital (SADH) and the San Francisco Kaiser Perma-nente Chemical Dependence Recovery Program, wereretrospectively divided into smokers (sALC, n=13)and non-smokers (nsALC, n=11). All were scannedtwice, 6±3 days after their last alcoholic drink atassessment point 1 (AP1) and 32±9 days after their lastdrink at assessment point 2 (AP2). The time betweenscans did not differ between groups (see Table 1).Fourteen healthy non-smoking, male light drinkers(nsLD) were recruited from the San Francisco Bay Areacommunity and scanned only once. All participants in thisstudy were examined as part of the correspondingspectroscopic, structural, and perfusion MRI studies(Durazzo et al., 2004, 2006; Gazdzinski et al., 2005,2006).

The inclusion and exclusion criteria are fullydescribed elsewhere (Durazzo et al., 2004). In summary,all ALC subjects met DSM-IV criteria for alcoholdependence with physiological dependence and con-sumed more than 150 standard alcoholic drinks permonth for at least 8 years prior to enrollment into the

Table 1Demographics and participant characteristics (mean±standard deviation)

Parameter ns

N

Age (years) 47Education (years) 16AMNART 11-yr average alcohol consumption (drinks/month)3-yr average alcohol consumption (drinks/month)Lifetime average alcohol consumption (drinks/month)Duration of drinking (years) 26Total lifetime alcohol consumption (kg)Onset of heavy drinking (years)Months of heavy drinkingTime after last alcoholic drink (days) at AP1Time after last alcoholic drink (days) at AP2Fagerstrom scoreCigarettes per dayDuration of smoking (years)Pack-yearsEthnicity

CaucasianAfrican-AmericanLatinoNative AmericanAsianPacific Islander/Polynesian

AMNART = American National Adult Reading Test; 1-yr average alcohol conaverage alcohol consumption = number of drinks per month over 3 years priormonth over lifetime; Duration of alcohol drinking = number of years of regdrink/month. Total lifetime alcohol consumption = total amount of pure EtOalcohol consumption exceeded 100 drinks per month. Pack years = (number of

study. A standard drink contains 13.6 g of pure ethanol,equivalent of 12 oz. beer, 5 oz. wine, or 1.5 oz. liquor.All participants were free of general medical, neurolog-ic, and psychiatric conditions, except unipolar mooddisorders, hypertension, and hepatitis C in ALCsubjects. Unipolar mood disorders were not exclusion-ary in ALC subjects due to their high reported incidenceamong alcohol-dependent individuals (e.g., Gilman andAbraham, 2001) and chronic cigarette smokers (e.g.,Fergusson et al., 2003).

Participants completed structured clinical interviewsas previously described (Durazzo et al., 2004). Two sALCand two nsALC subjects met DSM-IV criteria forsubstance-induced (alcohol) mood disorder with depres-sive features, and one nsALC subject was diagnosed withrecurrent major depression with mood-congruent psy-chotic symptoms. Exclusion of these participants fromanalyses did not affect the results. One nsALC subject metcriteria for past methamphetamine dependence, whereasone sALC subject met criteria for past opioid dependencewith physiologic dependence. However, both individualswere in sustained full remission, with last use 5 or more

LD nsALC sALC

=14 N=11 N=13

.3±8.2 50.2±9.1 50.7±9.0

.4±2.6 14.0±2.6 13.9±1.321±8 109±11 114±710±11 387±178 425±18710±11 385±179 366±14017±16 193±129 271±100.0±8.1 33.5±9.3 33.4±9.974±68 1051±818 1445±594– 26.6±9.9 21.8±3.7– 225±106 308±95– 5.4±2.7 6.4±3.3– 34.3±8.1 30.8±9.2– – 6.1±1.9– – 23±10– – 25±12– – 31±21

10 8 91 0 30 2 00 1 12 0 01 0 0

sumption = number of drinks per month over 1 year prior to study; 3-yrto study; Lifetime average alcohol consumption = number of drinks perular alcohol consumption (defined as consuming at least one standardH (kg) consumed over lifetime. Onset of heavy drinking = age, whencigarettes per day /20)× (duration of smoking at current level in years).

Fig. 1. Position of spectroscopic volume of interest overlaid on sagittalbrain scout image.

Fig. 2. Exemplary spectrum: one experimental spectrum with fittedbaseline (upper panel) and fitted spectrum overlaid on experimentalspectrum after subtraction of the fitted baseline (lower panel).

136 S. Gazdzinski et al. / Psychiatry Research: Neuroimaging 162 (2008) 133–145

years before enrollment. One participant in each ALCsubjects group had (medication-controlled) hypertension.Standard clinical laboratory examinations and a briefneurocognitive batterywere completedwithin 1 day of theMR studies. They assessed hepatocellular injury, redblood cell status, and visuospatial learning and memory(Brief Visual Memory Test-Revised; BVMT-R) (Bene-dict, 1997). Participants were allowed to smoke adlibitum prior to and during cognitive evaluation.

Alcohol consumption and smoking behavior over thelifetime were assessed via the Lifetime Drinking History(LDH; Skinner and Sheu, 1982; Sobell et al., 1988;Sobell and Sobell, 1992) and the Fagerstrom ToleranceTest for Nicotine Dependence (Fagerstrom et al., 1991),respectively (see Table 1). All sALC subjects continuedto smoke at their baseline levels during the assessmentinterval, except for one individual, who stoppedsmoking and used a nicotine patch; all his MR measuresimproved after AP1 but were within the range of thesALC group data. Six nsALC subjects never smokedand five had stopped smoking more than 5 years prior toenrollment. These never-smokers and past smokers didnot differ significantly on any of the neurocognitive orMR-derived measures at either AP. Twenty-one of 24ALC participated in continued outpatient substanceabuse treatment programs at the San Francisco VAMedical Center for the study duration; they receivedrandomly administer breathanalysis and were given

weekly drug screens to assure abstinence. The Institu-tional Review Boards of the University of CaliforniaSan Francisco and the San Francisco VAMedical Centerapproved all procedures, and informed consent wasobtained from all participants prior to study.

2.2. Data acquisition and processing

Data were acquired on a standard 1.5T MR system(Siemens Vision, Iselin, NJ). For AP1, hippocampi wereoutlined using a semi-automated high dimensional brainwarping algorithm (Medtronic Surgical NavigationTechnologies, Louisville, CO; Hsu et al., 2002) on T1-w magnetization-prepared rapid gradient echo imagesacquired with TR/TE/TI=10/7/300 ms, 15° flip angle,1×1 mm2 in-plane resolution, and 1.5-mm thick coronalpartitions oriented orthogonal to the long axes of hip-pocampi. Images at AP2 were coregistered to images atAP1 to assure use of the same landmarks forhippocampal delineation (see Hsu et al., 2002) at bothAPs. This approach was validated using 20 elderlyparticipants scanned about 1 year apart, and ityielded results similar to those obtained by independenthippocampal delineation for baseline and follow-upscans. Probability maps of GM, WM, and CSF withinmajor lobes, subcortical nuclei, brainstem, and cerebel-lum (but not hippocampi) were obtained from T1-w images by combining (1) three-tissue probabilisticsegmentation and (2) masks of major lobes, subcorticalnuclei, brainstem, and cerebellum. The latter structures

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were outlined on individual scans with a deformableregistration method that used an MRI atlas from a single36-year old control that had been manually divided intothe aforementioned structures (Cardenas et al., 2005).To correct for inter-participant variability in head size,absolute hippocampal volumes were scaled to totalintracranial volume (defined as sum of GM, WM, andCSF determined by segmentation).

A 1H MRSI dataset was acquired with TR/TE=1800/25 ms with PRESS pre-selection of a 100×60×15 mm3

volume of interest (VOI; Schuff et al., 1999). The VOIwas placed parallel to the long axes of the hippocampi andpositioned on the axial plane to cover both hippocampi(see Fig. 1). The MRSI field of view was 210×210 mm2

and was sampled using a circular k-space schemeequivalent to a maximum of 24×24 phase encodingsteps, resulting in a nominal and actual voxel resolution of1.1 ml and 1.6 ml, respectively (Schuff et al., 2001b).Post-processing details were described inMeyerhoff et al.(2004) and Schuff et al. (2001a). In short, integrals of theresonances corresponding to NAA, Cho, Cr, and m-Inowere estimated including baseline correction (see Fig. 2),adjusted for inter-participant differences in coil loading,transmitter voltage, the known SI point spread function,RF pulse profiles, and extrapolated to 100% tissue usingfractional tissue contributions to individual voxels thatwere obtained from aligned GM, WM, and CSF pro-bability maps. The final outcome measures were tissue-specific absolute metabolite concentrations expressed ininstitutional units; they were not reported inmolar units toavoid possibly inaccurate assumptions about relaxationtimes. For statistical analyses, we used only those voxelsthat passed our spectral quality assurance test as describedin Meyerhoff et al. (2004) and contained less than 25%CSF. MTL voxels contained at least 20% of hippocampaltissue and generally covered the body of the hippocam-pus. The average volume contributions to MTL SI voxelswere 31±3% from hippocampi, 32±5% from temporalWM, and 29±5% from temporal GM, without significantdifferences between groups.

2.3. Study design and statistical analyses

The nsLD group was scanned only once and wasused in cross-sectional comparisons of hippocampalvolumes and MTL metabolite concentrations withsALC and nsALC subjects at 1 week and 1 month ofabstinence. These cross-sectional assessments utilizedanalysis of covariance (ANCOVA), with age as thecovariate, and followed up with pair-wise contrasts. Thelongitudinal analyses evaluated hippocampal volumeand metabolite concentrations only in sALC and nsALC

subjects with repeated measures ANOVAs. The within-subject factor (AP) examined longitudinal changes inhippocampal volumes and absolute metabolite concen-trations across APs in the combined ALC (i.e., nsALC+sALC) group. The AP-by-smoking status (between-subjects factor) interaction evaluated potential differ-ences in recovery of hippocampal volumes and absolutemetabolite concentrations between sALC and nsALCsubjects.

Additionally, we examined rates of change forhippocampal volumes and MTL metabolite concentra-tions for sALC and nsALC subjects using independentt-tests. Change rates were defined as:

Rate of Change ¼MeasureðAP2Þ �MeasureðAP1Þ

MeasureðAP1ÞdðTime between AP2 and AP1 in monthsÞ d100%

Hippocampal volumes and MTL metabolites wereseparately correlated with seven drinking and foursmoking variables, three measures of depressive,withdrawal and anxiety symptomatology, nine labora-tory variables, and two neurocognitive measures. Toaccount for multiple comparisons, the alpha levels forthe corresponding families were set to 0.007 (=0.05/7),0.012, 0.017, 0.006, and 0.025, respectively. Relation-ships were assessed with Spearman correlations. Allstatistical tests were conducted with SPSS-12.0 forWindows (SPSS; Chicago, IL).

3. Results

3.1. Participant characterization

Groups were matched on age [F(2,35)=0.62, P=ns],but nsLD had more education than sALC and nsALC [F(2,35)=5.22, P=0.01]. The sALC and nsALC subgroupshad a similar average number of drinks per monthconsumed over 1 and 3 years prior to enrollment (P=0.6).However, sALC subjects tended to begin drinking atlevels higher than 100 drinks/month at a younger age(P=0.06) and tended to have more drinks per month overthe lifetime (P=0.10) than nsALC subjects. Detaileddemographics and participant characteristics for allgroups are given in Table 1. The sALC group did notdiffer from the nsALC group at any AP on self-reportmeasures of depressive, anxiety, withdrawal symptom-atology or most laboratory variables. However, gamma-glutamyltransferase and aspartate aminotransferase levelsin the nsALC group at AP1 were elevated but normalizedby AP2 (see Table 2). The measures of withdrawalsymptomatology for the sALC and nsALC groups at AP1were not clinically elevated. The sALC Fagerstrom score

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was 6.1±1.9 (min=2, max=10), indicating a medium tohigh level of nicotine dependence.

3.2. Right–left hemisphere comparisons

There were no significant group differences inhippocampal volumes and metabolite concentrationsbetween right and left hemispheres in this cohort. Thus,bilateral hippocampal volumes and metabolite concen-trations averaged over both hemispheres are reported.

3.3. Cross-sectional group comparisons at assessmentpoint 1

3.3.1. Hippocampal volumesAn ANCOVA comparing hippocampal volume be-

tween the nsLD, nsALC and sALC groups was notsignificant [F(2,35)=1.85, P=0.17]. However, amongthe hypothesized contrasts, sALC volumes were 7.8%smaller than nsALC volumes (P=0.05) and tended to be6.9% smaller than in the nsLDgroup (P=0.08; see Fig. 3).No significant volume differenceswere observed betweenthe nsALC and nsLD groups.

3.3.2. MTL metabolite concentrationsFor NAA, the groups were significantly different [F

(2,34)=3.77, P=0.03], with 10.4% lower concentrationin the sALC group (P=0.02) and 12.8% lower concen-

Table 2Depressive, anxiety, and withdrawal symptomatology and laboratory variabl

Parameter nsLD nsALC

AP1

BDI 3.9±4.0 15.5±9.7STAI Y-2 33±8 48±11CIWA-Ar – 3.5±3.7GGT (i.u.) 28±18 129±16AST (i.u.) 27±8 45±47ALT (i.u.) 30±13 59±55Albumin (g/dl) 4.1±0.3 3.9±0.3Prealbumin (mg/dl) 31.4±6.3 27.9±7.1WBC 6.3±1.5 7.2±1.3RBC 4.90±0.37 4.60±0.4Hemoglobin (g/dl) 15.1±1.1 14.7±1.6Hematocrit (%) 43.9±3.2 42.6±3.7MCV 89.5±2.9 91.1±6.7Hep-C (number of participants) 0 2

BDI = Beck Depression Inventory; STAI Y-2 = State-Trait Anxiety Inventoryof Withdrawal Assessment for Alcohol; GGT = gamma-glutamyltransferaaminotransferase; local normal range = 5–35 institutional units; ALT = alaAlbumin local normal range = 3.3 – 5.2 g/dl; Prealbumin local normal range4.8–10.8 K/mm3; RBC = red blood cell count; local normal range = 4.7–6.1 Mnormal range = 42–52%.

tration in the nsALC group (P=0.008) compared with thensLD group (see Fig. 3). For Cho, there was a trend forgroup differences [F(2,34)=3.05, P=0.06]. Among thehypothesized contrasts, both the nsALC and sALCgroupsdemonstrated 12% lower Cho concentrations thanthe nsLD group (P=0.03 and P=0.02, respectively;see Fig. 3). NAA and Cho concentrations were not signi-ficantly different between the sALC and nsALCgroups. A trend for group differences was apparent form-Ino [F(2,34)=2.98, P=0.06] (see Fig. 3), but no signi-ficant group differenceswere found forMTLCr [F(2,34)=0.44, P=0.69]. Upon co-variation for inter-participantdifferences in hippocampal tissue contributions to MTLvoxels, the reported results remained virtually unchanged.

3.4. Longitudinal volumetric and metabolite changesfor the nsALC and sALC groups

3.4.1. Hippocampal volumesThere was a significant main effect for AP (i.e., time)

[F(1,22) = 7.51, P = 0.01], indicating significantincreases of hippocampal volume in the ALC group(i.e., sALC+nsALC) over approximately 1 month ofabstinence from alcohol (see Fig. 3). The interactionbetween smoking status and AP was not significant [F(1,22)=0.02, P=0.96], consistent with similar rates ofchange of hippocampal volume observed for the sALCand nsALC groups (3.6±5.8% vs. 3.4±6.4%, P=0.48).

es by group and assessment point (AP) (mean±standard deviation)

sALC

AP2 AP1 AP2

5.7±5.9 15.6±10.3 11.8±10.643±10 50±13 44±130.1±0.3 2.5±2.8 0.5±1.3

4 59±76 61±35 48±3224.8±7.5 33±9 31.0±12.0

– 28±7 –4.1±0.4 4.0±0.2 4.7±2.324.7±7.0 26.7±4.1 27.9±4.17.2±1.9 6.6±2.0 8.0±1.9

8 4.76±0.35 4.27±0.32 4.55±0.4215.0±1.4 13.8±0.9 14.4±1.043.1±4.2 40.6±2.4 42.3±2.990.4±5.8 95.1±3.7 93.1±3.7

2 2 2

— State; CIWA-Ar = Addiction Research Foundation Clinical Institutese; local normal range = 7–64 institutional units; AST = aspartatenine aminotransferase; local normal range = 7–56 institutional units;= 18–45 mg/dl; WBC = white blood cell count; local normal range =/mm3; Hemoglobin local normal range = 14–18 g/dl; Hematocrit local

139S. Gazdzinski et al. / Psychiatry Research: Neuroimaging 162 (2008) 133–145

3.4.2. MTL metabolite concentrationsThere were significant interactions between smoking

status and AP for NAA [F(1,22)=4.25, P=0.05] and Cho[F(1,22)=6.29,P=0.02] (Fig. 3). Comparedwith findingsat AP1, nsALC subjects had higher Cho at AP2 (P=0.01)and tended to have higher NAA at AP2 (P=0.06). InsALC subjects, there were no longitudinal changes inNAA and Cho. Correspondingly, sALC compared withnsALC subjects had significantly smaller rates of changein NAA (−3±26% vs. 12±15%,P=0.05) and Cho (−1±31% vs. 19±19%, P=0.04). The interactions anddifferences in rates of change for Cr and m-Ino were notsignificant. Also, nomain effects for APwere observed forany metabolite [F(1,22)b2.57, PN0.12].

3.5. Cross-sectional group comparisons at assessmentpoint 2

3.5.1. Hippocampal volumesNo significant group differences were observed for

hippocampal volume at AP2 [F(2,34)=1.42, P=0.26].Among hypothesized contrasts, sALC subjects had 7.7%smaller hippocampi than nsALC subjects (P=0.05).

Fig. 3. Baseline values and recovery of hippocampal volumes, scaled to intra(lower left), and MTL — m-Ino (lower right). Non-smoking and smoking arespectively. Range of measures in nsLD subjects is depicted in gray (mean

3.5.2. MTL metabolite concentrationsAt AP2, significant group differences were seen for

Cho [F(2,34)=5.41, P=0.009] with a trend toward adifference for NAA [F(2,34)=2.80, P=0.075]. Cho insALC subjects was 14.3% lower than in nsLD subjects(P=0.007) and 16.7% lower than in nsALC subjects(P=0.003). NAAwas 12.3% lower in sALC subjects thanin nsLD subjects (P=0.02) and tended to be 10.5% lowerin sALC than nsALC subjects (P=0.06). Corresponding-ly, NAA and Cho were similar in nsALC and nsLDsubjects (PN0.40). Neither Cr nor m-Ino differedsignificantly between groups (PN0.59). Covarying forinter-participant differences in hippocampal tissue con-tributions to MTL voxels did not change the reportedlevels of significance appreciably.

3.6. Correlations among outcome measures

3.6.1. Hippocampal volumesYounger age of onset of heavy drinking in com-

bined ALC (i.e., nsALC+sALC) subjects correlatedwith larger hippocampal volumes at both AP1 andAP2 (rho≤0.51, Pb0.008). This correlation was more

cranial volume (upper left), MTL — NAA (upper right), MTL — Cholcohol-dependent groups are depicted with open and closed symbols,±standard error).

140 S. Gazdzinski et al. / Psychiatry Research: Neuroimaging 162 (2008) 133–145

pronounced in nsALC subjects and remained significantafter controlling for age and after eliminating fromanalyses all participants with depressive disorders. Higheraverage number of drinks per month over the lifetime wasassociated with larger hippocampal volumes at both APsin nsALC (rhoN0.89, Pb0.001, N=11), but not in sALC(|rho|b0.22, PN0.44) subjects. No correlations betweenhippocampal volumes and measures of smoking severityreached the level of significance. In nsALC subjects over1 month of abstinence, improving visuospatial memorytended to correlate with increasing hippocampal volumes(rho=0.60, P=0.03); this relationship was not apparent insALC subjects.

3.6.2. MTL metabolite concentrationsBetween AP1 and AP2, in the combined ALC

group, improving visuospatial memory correlated withincreases of MTL Cho, Cr, and m-Ino (rhoN0.40,Pb0.025). These correlations were similar in sALC andnsALC subjects (see Fig. 4). In nsALC, but not in sALCsubjects, a larger m-Ino increase tended to correlate withgreater average number of drinks per month over thelifetime and earlier onset of heavy drinking (|rho|N0.63,Pb0.04), whereas a larger Cr increase tended to beassociated with greater average number of drinks permonth over the lifetime (rho=0.62, P=0.04). At AP2,but not at AP1, better visuospatial memory correlatedwith higher MTL NAA in the combined ALC group(rho=0.43, P=0.02). These relationships were similarin sALC and nsALC subjects. Metabolite concentrationsand their changes were not related to measures ofdrinking or smoking severity after correction formultiple comparisons. Furthermore, MTL metaboliteconcentrations and their changes did not correlate withhippocampal volumes and their changes.

Fig. 4. Significant correlation between change in concentration ofcholine-containing metabolites and visuospatial memory over the firstmonth of abstinence from alcohol (rhoN0.40, Pb0.025).

4. Discussion

These preliminary MR studies in abstinent alcohol-dependent men describe hippocampal volumes andmedial temporal lobe metabolite concentrations at 1week of abstinence from alcohol and their changes overapproximately 1 month of sobriety. At 1 week ofabstinence, we observed smaller hippocampal volumesin sALC than in nsALC subjects, but no volumereductions in nsALC relative to nsLD subjects; however,both sALC and nsALC subjects demonstrated signifi-cantly lower MTL NAA and Cho compared with nsLDsubjects. Over 1 month of abstinence from alcohol, bothsALC and nsALC subjects demonstrated similar magni-tudes of hippocampal volume increases, but sALCsubjects continued to exhibit smaller volumes thannsALC subjects at 1 month of sobriety (i.e., AP2). Overthe same period, NAA and Cho concentrations did notsignificantly change in sALC subjects and, at 1 month ofabstinence, sALC subjects continued to demonstratelower NAA and Cho than seen in both nsALC andnsLD subjects. In contrast, MTL NAA and Choconcentrations normalized in nsALC subjects. Improve-ments in visuospatial memory were associated withincreases of hippocampal volumes in nsALC subjectsonly and with changes of Cho, Cr, and m-Ino in bothsALC and nsALC subjects. At 1 month of sobriety, bettervisuospatial memory in nsALC subject correlated withhigher NAA concentrations. Inasmuch as these correla-tions were essentially not observed in other brain regionsof a similar cohort (Durazzo et al., 2006), the associationssuggest that the changes in hippocampal volumetric andMTL spectroscopic measures are functionally significantand reflect the integrity of MTL tissue, includingpathways subserving memory and other related cognitiveabilities.

4.1. Hippocampal volumes

Hippocampal volume reductions were observed inrecently detoxified alcoholics (Bleich et al., 2003).However, in our study, only sALC subjects at 1 week ofabstinence showed smaller hippocampi than bothnsALC and nsLD subjects, whereas nsALC subjectsdid not significantly differ from nsLD subjects on thismeasure. This is consistent with our earlier report ofsmaller lobar GM volumes in sALC subjects than inboth nsALC and nsLD subjects (Gazdzinski et al.,2005). The significant hippocampal volume increaseover the first month of abstinence is consistent withvolumetric recovery observed in other brain regionsduring sobriety (e.g., Pfefferbaum et al., 1995;

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Gazdzinski et al., 2004). It suggests that previous cross-sectional studies that measured hippocampal volumes atabout 1 month of abstinence (Agartz et al., 1999; Laaksoet al., 2000; Sullivan et al., 1995) did not capture the fullextent of (i.e., underestimated) chronic alcohol-associatedhippocampal volume deficits. The mechanisms ofhippocampal volume reductions at 1 week of abstinenceand subsequent volume increase in both sALC andnsALC subjects may involve dearborization and rear-borization of hippocampal dendrites (King et al., 1988;Lescaudron et al., 1989; Cadete-Leite et al., 1988; Durandet al., 1989; McMullen et al., 1984) or generalizedchanges in the neuropil of this structure. Although smallhuman neuropathological studies did not show frankneuronal loss in hippocampi of alcoholics, they did notaccount for smoking status of the participants; thus,hippocampal neuron loss in smokers cannot be excluded.Cigarette smoke contains many toxic compounds in thegas and particulate phases [e.g., carbon monoxide, freeradicals, nitrosamines, polynuclear aromatic compounds(Fowles et al., 2000)] that may directly or indirectlycompromise central nervous system tissue and vascularendothelial function (for review, see Durazzo et al., 2006).The chronic exposure of sALC subjects to thesepotentially noxious compounds in cigarette smoke maycontribute to the smaller volumes observed in sALCsubjects relative to both nsALC and nsLD subjects at AP1(∼8%), as well as the continued volume differencesobserved between sALC and nsALC subjects at AP2(∼8%).

The unexpected positive relationships between largerhippocampal volumes and earlier onset of heavydrinking in both sALC and nsALC subject, as well asassociations between larger hippocampal volumes andmore drinks per month over the lifetime in nsALCsubject were not apparent in our previous investigationsof other brain regions in a similar patient cohort(Gazdzinski et al., 2005). We do not have a clear-cutexplanation for these associations. They may reflect theinfluence of factors other than alcohol on hippocampalsize such as psychosocial stress (Smith, 1996; Winterand Irle, 2004), mood disorders (Campbell and Macqu-een, 2004; Videbech and Ravnkilde, 2004), personalitydisorders (Laakso et al., 2000), withdrawal-inducedneurotoxicity (Crews et al., 2005; Prendergast et al.,2000), genetic susceptibility (Hill et al., 2006), or themodest size of our cohort.

4.2. MTL metabolite concentrations

Decreased NAA and Cho concentrations in MTL at1 week of abstinence in both sALC and nsALC subjects

are consistent with metabolite abnormalities reported inother brain regions of alcohol-dependent individualsduring early abstinence (Durazzo et al., 2004; Endeet al., 2005; Parks et al., 2002; Seitz et al., 1999). In ourcorresponding 1H MRSI study (Durazzo et al., 2004),which assessed metabolite concentrations in lobar, sub-cortical, and cerebellar brain regions (but not inhippocampus) in many 1-week-abstinent alcoholics alsostudied for this report, we found that chronic smokingwasassociated with lower NAA and Cho levels in the frontallobe and several subcortical structures. However, sALCand nsALC subjects did not differ from each other ornsLD subjects on temporal lobe WM or GM NAA andCho levels.

No significant longitudinal changes in MTL NAA,Cho, or m-Ino levels were observed in sALC subjects,whereas all MTL metabolite concentrations were nor-malized in nsALC subjects after 1 month of sobriety.These different patterns of recovery between sALC andnsALC subjects are consistent with our longitudinal 1HMRSI findings in recovering alcoholics (Durazzo et al.,2006). The absence of longitudinal NAA and Choincreases in sALC subjects, again, may reflect the directand indirect adverse effects of continued exposure tocigarette smoke on brain tissue integrity and vascularendothelial function (discussed in more detail in Durazzoet al., 2006). Additionally, nicotine administration in ratsinhibits dentate gyrus neurogenesis (Abrous et al., 2002;Shingo and Kito, 2005), which otherwise is increasedduring withdrawal from alcohol (Nixon and Crews,2004). Since the dentate gyrus constitutes only about3% of human hippocampal volume (Harding et al., 1998),it is not likely that neurogenesis contributes significantlyto hippocampal volume changes with abstinence fromalcohol. However, the dentate gyrus is part of theessentially tri-synaptic network of the hippocampus andthe addition of relatively small numbers of new neurons inthis structure may lead to relatively large differences insignal processing (Kempermann et al., 2004), potentiallyleading to larger synaptic and metabolic activity inhippocampus/MTL, and thus to increased NAA levels(Meyer-Lindenberg et al., 2005). This process may alsobe associated with cell membrane changes reflected in theobserved Cho increases.

Although the sALC group did demonstrate hippocam-pal volume increases over 1 month of abstinence, thelack of significant MTL metabolite recovery maysuggest incomplete recovery of hippocampal and associ-ated tissue integrity in sALC subjects. Conversely,the longitudinal NAA increases observed in nsALCsubjects suggest greater recovery of neuronal structuralelements or metabolism, while Cho increases may

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indicate normalization of parenchymal cell membranesynthesis/turnover or recovery of para-hippocampalcortex and myelin (Harding et al., 1997; Martin et al.,1995). Previous studies in abstinent alcoholics did notevaluate smoking effects, although the samples presum-ably included a significant number of smokers; thesestudies generally demonstrated little or no NAA recoveryin major lobes and cerebellum over the first month ofabstinence (Ende et al., 2005; Parks et al., 2002, but seeBendszus et al., 2001). Finally, lowerMTLNAA in sALCsubjects at 1 month of abstinence compared with nsALCand nsLD subjects is consistent with smoking-relatedhippocampal NAA reductions in non-alcoholic popula-tions (Gallinat et al., 2007).

4.3. Functional significance

Over the first month of abstinence, MTL metaboliteconcentration increases in nsALC and sALC subjectswere related to improvements of visuospatial learning.These neurobiological changesmay reflect rearborization,axonal regrowth, and/or neurogenesis and may havefunctional significance. For example, studies in ratsdemonstrated that the number of new cells generatedin the dentate gyrus was associated with better perfor-mance on hippocampus dependent learning (Eisch, 2002).The fact that Cho, Cr, and m-Ino increases correlatewith improvements of visuospatial memory in alcoholicsover the first month of abstinence suggests that MTLcell membranes and myelin integrity play important rolesin facilitating performance on hippocampus-mediatedtasks.

Our participants were allowed to smoke ad libitumprior to and during neurocognitive assessment. However,we do not believe nicotine-related cognitive enhancementor withdrawal significantly confounded our cognitive testresults. In healthy non-smokers and in individuals withattention deficit hyperactivity disorder and schizophrenia-spectrum disorders, acute nicotine administration tran-siently improves some areas of neurocognition, mostappreciably sustained attention (see Rezvani and Levin,2001; Sacco et al., 2004). Whether these acute effects onneurocognition are alsomanifested in alcoholics and othersubstance abusers is unclear (see Ceballos et al., 2005,2006). The adverse effects of nicotine withdrawal onneurocognition are not typically apparent until 8–12 hafter the last nicotine dose, at least in non-alcoholicchronic smokers (see Sacco et al., 2004; Mendrek et al.,2006; Xu et al., 2005). This is likely attributable to themaintenance of relatively high levels of plasma nicotinedue to repeated smoking during waking hours (Hukkanenand Benowitz, 2005).

4.4. Limitations

MTL 1H MRSI voxels contain contributions fromhippocampi and surrounding WM and GM, making itimpossible to determine the affected tissue type. Explor-atory analyses not reported here, however, showed thatmetabolite concentrations in temporalWMdid not changesignificantly over time in either sALC or nsALC subjects.Therefore, the observed MTL metabolite changes likelyoriginate in GM of the hippocampi and/or temporalcortex. The group sizes in this study were modest, as weincluded only those participants who had completedatasets at both AP1 and AP2. All cross-sectional results,however, remained unchanged when we analyzed about40% larger groups at 1 week of abstinence (includingthose who did not haveAP2) and at 1month of abstinence(including those who were enrolled between AP1 andAP2). Including only male participants in the analysesprecluded assessment of sex effects. Finally, potentialunrecorded group differences in nutrition, stress, exercise,overall physical health, and genetic predispositions maycontribute to the results described in this study.

4.5. Conclusions

Hippocampal volume and recovery of MTL meta-bolites that reflect neuronal viability and membranesynthesis/turnover in abstinent alcohol-dependent menappear to be adversely affected by comorbid chroniccigarette smoking. MTL spectroscopic measures andtheir changes may be functionally significant as theycorrelate with performance and improvement of hippo-campus-mediated cognitive tasks. Our previous MRstudies assessing regional lobar brain metabolites, tissuevolumes, and perfusion in recovering alcoholics suggestthat chronic cigarette smoking compounds aspects ofalcohol-induced brain injury and modulates neurobio-logical recovery during short-term abstinence. Ifreplicated, this research collectively will give supportto the growing clinical movement of encouragingchronic smokers entering treatment for alcohol usedisorders to consider concurrent participation in asmoking-cessation program. On a more general note,our neuroimaging results may also be relevant to otherconditions with high smoking co-morbidity and estab-lished hippocampal/MTL involvement, such as poly-substance abuse, schizophrenia, and mood disorders.The long-term effects of cigarette smoking likely opposethe acute and positive effects of nicotine on attention,learning, and memory, by imparting biological injury onbrain tissue and ultimately resulting in functionalcompromise and possibly poorer long-term outcome.

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Acknowledgements

NIH AA10788 (DJM) supported this project. Wethank Mary Rebecca Young, Bill Clift and Dr. DonaldTusel of the San Francisco VA Substance Abuse DayHospital, Dr. David Pating, Karen Moise and theircolleagues at the San Francisco Kaiser PermanenteChemical Dependency Recovery Program for theirvaluable assistance in recruiting research participants.We also thank Diana Truran and Derek Flenniken forassistance with data analyses and Erin Clevenger andDaniel Rosenbaum for hippocampal volume analyses.

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