Smaller amygdala volume and reduced anterior cingulate gray matter density associated with history of post-traumatic stress disorder

Psychiatry Research: Neuroimaging 174 (2009) 210–216

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Psychiatry Research: Neuroimaging j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p s yc h r e s n s

Smaller amygdala volume and reduced anterior cingulate gray matter density associated with history of post-traumatic stress disorder Mark A. Rogersa,⁎, Hidenori Yamasueb, Osamu Abec, Haruyasu Yamadac, Toshiyuki Ohtanib, Akira Iwanamid, Shigeki Aokic, Nobumasa Katod, Kiyoto Kasaib a

Department of Psychology, Monash University, Clayton 3800, Victoria, Australia Department of Neuropsychiatry, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Department of Radiology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan d Department of Neuropsychiatry, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-8666, Japan b c

a r t i c l e

i n f o

Article history: Received 2 October 2007 Received in revised form 23 February 2009 Accepted 4 June 2009 Keywords: PTSD MRI Amygdala Fear extinction

a b s t r a c t Although post-traumatic stress disorder (PTSD) may be seen to represent a failure to extinguish learned fear, significant aspects of the pathophysiology relevant to this hypothesis remain unknown. Both the amygdala and hippocampus are necessary for fear extinction occur, and thus both regions may be abnormal in PTSD. Twenty-five people who experienced the Tokyo subway sarin attack in 1995, nine who later developed PTSD and 16 who did not, underwent magnetic resonance imaging (MRI) with manual tracing to determine bilateral amygdala and hippocampus volumes. At the time of scanning, one had PTSD and eight had a history of PTSD. Results indicated that the group with a history of PTSD had significantly smaller mean bilateral amygdala volume than did the group that did not develop PTSD. Furthermore, left amygdala volume showed a significant negative correlation with severity of PTSD symptomatology as well as reduced gray matter density in the left anterior cingulate cortex. To our knowledge, this is the first observation of an association between PTSD and amygdala volume. Furthermore the apparent interplay between amygdala and anterior cingulate cortex represents support at the level of gross brain morphology for the theory of PTSD as a failure of fear extinction. Crown Copyright © 2009 Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Post-traumatic stress disorder (PTSD) is perhaps the most lifedisrupting of the anxiety conditions, with markedly adverse effects on occupational and social functioning as well as increased risk of suicidal behaviour (Beckham et al., 1996). PTSD has been reported to be associated with a number of neuropathological signs, including the following: abnormal serotonin (Southwick et al., 1999) and noradrenergic (Kosten et al., 1987; Perry et al., 1987) function, and dysregulation of the hypothalamic-–pituitary–adrenal axis (Yehuda et al., 1995; Maes et al., 1998). The clinical nature of PTSD, in which a fear response subsequent to a traumatic event continues to be expressed in situations that are objectively safe and with no recurrence of trauma, and the effectiveness of therapies that closely mimic extinction procedures has led to the hypothesis that PTSD may be explained by a failure to appropriately extinguish learned fear responses (Myers and Davis, 2007). This hypothesis is supported by evidence at the neurophysiological level that similar processes are involved in failure to extinguish learned fear and in PTSD (reviewed in Rauch et al., 2006). In particular, ⁎ Corresponding author. Tel.: +61 3 99053976; fax: +61 3 9905 3948. E-mail address: [email protected] (M.A. Rogers).

there is evidence that the anterior cingulate cortex (ACC), the amygdala and the hippocampus (Bouton et al., 2006) are important in the processes involved in normal fear extinction (Myers and Davies, 2007; Herry and Garcia, 2002) and that these same regions show abnormal functioning in patients with PTSD (Shin et al., 2001). Successful extinction of learned fear depends upon activity of amygdala N-methyl-D-aspartate (NMDA) receptors (Myers and Davis, 2007) while decreased ACC activity following extinction appears to be associated with persistence of fear responses (Herry and Garcia, 2002). Thus, it may be that the ACC acts to facilitate fear extinction by modulating the amygdala and that dysfunction of this mechanism may result in failure to extinguish learned fear (Herry and Mons, 2004). Indeed, a number of functional imaging studies have suggested that excessive amygdala activity and reduced ACC activity are present in PTSD (reviewed in Rauch et al., 2006). Functional imaging studies employing symptom provocation and cognitive activation tasks have identified greater activation of the amygdala, anterior paralimbic structures, Broca's region, and other neocortical regions, and a failure of activation of ACC in response to traumarelated stimuli in individuals with PTSD (Pitman et al., 2001; Shin et al., 2001; Villarreal and King, 2001). In contrast to the functional imaging evidence of ACC and amygdala involvement in PTSD, there have been few reported studies of

0925-4927/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pscychresns.2009.06.001

M.A. Rogers et al. / Psychiatry Research: Neuroimaging 174 (2009) 210–216

structural abnormality of these regions in PTSD (Rauch et al., 2003; Yamasue et al., 2003; Corbo et al., 2005; Kitayama et al., 2006; Woodward et al., 2006). In terms of the ACC, Yamasue et al. (2003) identified reduced gray matter density of left hemisphere ACC in trauma victims with PTSD compared to those without PTSD and, Abe et al. (2006) identified increased fractional anisotropy in a subjacent region of the left ACC of the same sample. Efforts to identify structural abnormality of the amygdala in PTSD have been less successful. Compared to those studies that have measured hippocampal volume (reviewed in Geuze et al., 2005), few studies have examined amygdala volume (Gurvits et al., 1996; Bremner et al., 1997; Fennema-Notestine et al., 2002; Lindauer et al., 2004; Wignall et al., 2004). Indeed, previous studies, most of which reported smaller-than-normal hippocampal volume, have found no evidence of abnormal amygdala volume in patients with PTSD (reviewed in Rauch et al., 2006), although one study reported reduced amygdala volume associated with the presence of intrusive recollections in cancer survivors (Matsuoka et al., 2003). A recent study further showed a nonlinear association between the total lifetime number of traumatic episodes experienced and amygdala gray matter volume in healthy adults who had experienced the World Trade Center disaster on 9/11/01 (Ganzel et al., 2008). In contrast, no previous study reported regional volume enlargement in the amygdala of patients with PTSD. Taking this evidence into consideration, it seems that PTSD is more likely to be associated with reduced rather than increased amygdala volume. It may appear anomalous to predict reduced amygdala volume in conjunction with reports of increased amygdala activity, but in fact previous functional imaging studies have revealed abnormally increased activation in brain regions showing volume reduction in several neuropsychiatric disorders (e.g. schizophrenia: Callicott et al., 2000; major depression: Sheline et al., 2001). Most adult psychiatric disorders, including schizophrenia, depression, panic disorder, and PTSD, have not shown regional brain volume enlargement but rather regional brain volume reduction (Shenton et al., 2001; Geuze et al., 2005), although childhood psychiatric illness sometimes shows increased volume of the amygdala (e.g. Schumann et al., 2004). Reduced amygdala volume has also been reported in dissociative identity disorder (Vermetten et al., 2006) and Bipolar disorder (Berretta et al., 2007). It may be that there is no effect of reduced amygdala volume in PTSD. However, it is also possible that methodological factors have contributed to these negative results. For instance, early studies had insufficient image resolution (3 mm or more) to reliably delineate small subcortical structures (Bremner et al., 1997). Other studies defined a certain coronal slice, for example, the last slice before the appearance of mammillary bodies, as the anterior and posterior boundary of the amygdala (Gurvits et al., 1996; Bonne et al., 2001; Lindauer et al., 2004; Wignall et al., 2004). The method of tracing of the amygdala employed in pervious studies involved working only from coronal slices. However, this approach is not optimal, particularly for the anterior boundary of the amygdala and the boundary between the amygdala and the hippocampus, which are difficult to discern accurately from coronal slices. In comparison, the present study employed a significantly more accurate tracing method in which every slice was checked in each of three orthogonal planes (a detailed description of tracing methodology appears in Section 2) to assess amygdala volume in individuals who have experienced an acute traumatic event. Another potential moderating variable is illness chronicity, since animal studies have reported an increase of amygdala tissue due to chronic stress (Vyas et al., 2003; Mitra et al., 2005). A further potential source of confounding is medication with psychoactive agents and drug abuse. To date only a single study (Bossini et al., 2008) has employed a PTSD sample free of treatment for PTSD. The sample employed in the current sample had no history of treatment for PTSD. Indeed, 23 of the participants had never received any psychiatric

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treatment before participating in this study. Two of the nine victims determined to have had PTSD had received benzodiazepines for insomnia or general anxiety for 2–4 months from 2–4 years prior to participating in this study. This extremely light medication history may be a considerable advantage for the present study over past research. The present sample consists of 25 individuals who experienced the Tokyo subway sarin attack, nine of whom subsequently developed PTSD (PTSD-history group) and 16 who did not (No PTSD-history group). Those with a history of PTSD have previously been found to have reduced left ACC gray matter density relative to those with no history of PTSD (Yamasue et al., 2003) as well as increased fractional anisotropy subjacent to the same left ACC region (Yamasue et al., 2007). As effective extinguishing of learned fear involves interaction of the ACC and amygdala and as these patients have demonstrated left hemisphere ACC abnormality, it was predicted that left hemisphere amygdala volume would be reduced in proportion to the previously observed reduced gray matter density of the ACC in those participants with a history of PTSD relative to those with no history of PTSD. 2. Method 2.1. Participants Details of the participants have been provided previously (Yamasue et al., 2003). Briefly, 25 people (10 females), all of whom experienced the Tokyo subway sarin attack in 1995, participated in the study. Of these 25, nine were diagnosed with PTSD (1 man, current PTSD; 8 [4 men and 4 women], had a history of PTSD) while the remaining 16 had neither current PTSD nor a history of PTSD. Duration of the disorder ranged from 1 to 63 months (mean = 10.7 months; SD = 19.9). None of the participants had ever received treatment for PTSD. Three of the nine victims diagnosed as having had PTSD had psychiatric comorbidity: current major depression (n = 1), current (n = 1) and history (n = 1) of panic disorder with agoraphobia. The prevalence of psychiatric comorbidity did not significantly differ between groups (depression, P = 0.36; panic disorder, P = 0.12; Fisher's exact test). Although two of the nine subjects diagnosed with a history of PTSD had received benzodiazepines (for insomnia or general anxiety) for 2–4 months some 2–4 years prior to their participation, the other 23 subjects had never received psychiatric treatment before participating in this study. None of the 25 subjects had a history of neurological illness, serious head trauma with any known cognitive consequences or loss of consciousness for more than 5 min, or alcohol/substance abuse or dependence. None of the 16 victims without PTSD-history had any history of neuropsychiatric disorder or a family history of axis I disorder in their first-degree relatives. The subjects with a history of PTSD had significantly higher mean IES score than those without PTSD-history (PTSD-history: mean = 25.22 S.D. = 7.21; No PTSD-history: mean = 11.88, S.D. = 10.90). Diagnostic interviews and magnetic resonance imaging (MRI) acquisition were conducted on the same day, which was 5–6 years after the sarin attack. Participants completed the revised Impact of Event Scale (Asukai et al. 2002) and the Japanese version of the Clinician-administered PTSD scale (CAPS) (Asukai and AshizonoMaher, 1998), and were screened for neuropsychiatric disorder using the Mini International Neuropsychiatric Interview (MINI) (Sheehan et al., 1998). Demographic information is given in Table 1. 2.2. MRI acquisition The method of 1.5-mm slice, high spatial resolution MRI acquisition was the same as that employed in our previous studies (Yamasue et al., 2003; 2004). Briefly, the MRI data were obtained using a 1.5-

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M.A. Rogers et al. / Psychiatry Research: Neuroimaging 174 (2009) 210–216

Table 1 Demographic characteristics of study participants. Victims with PTSD (n = 9)

Victims without PTSD (n = 16)

t-test

Variables

Mean

S.D.

Mean

S.D.

t-value

P

Age SESa Parental SESa Education (years) IES scores CAPS scores Total Hyperarousal Avoidance Re-experiencing

44.56 2.56 3.11 12.78 25.22

15.96 1.01 0.93 1.99 7.21

44.44 2.13 2.98 13.75 11.88

13.60 0.81 0.68 2.21 10.90

0.02 1.17 0.37 − 1.01 − 3.28

n.s. n.s n.s. n.s 0.003

23.89 10.78 7.67 5.41

11.03 6.08 4.53 3.71

5.38 4.00 0.94 0.04

5.41 4.27 1.70 0.96

5.67 3.27 5.38 5.17

0.000 0.003 0.000 0.000

The degrees of freedom were [1,23] for all statistics. a Socioeconomic status, assessed using the Hollingshead-Relich scale. Higher scores indicate lower status.

Tesla scanner (General Electric Signa Horizon Lx version 8.2, GE Medical Systems, Milwaukee, WI, USA). Three-dimensional Fouriertransform spoiled gradient recalled acquisition with steady state was used because it affords excellent contrast between gray matter and white matter in the evaluation of brain structures. The repetition time was 35 ms, the echo time 7 ms with one repetition, the nutation angle was 30°, the field of view 24 cm, and the matrix 256 × 256 (192) × 124. A trained neuroradiologist (Ha.Ya. or O.A.) evaluated the MRI scans and found no gross abnormalities in any of the subjects. 2.3. Manual tracing for hippocampus and amygdala volumetry The amygdala and hippocampus gray matter regions of interest (ROIs) were outlined manually by one rater (M.A.R.) blind to diagnostic status. For the manual tracing, we used a software package for medical image analysis (3D Slicer; software available at http://www.slicer.org), which enables a simultaneous view of orthogonal planes. The landmarks used to delineate the ROIs in the present study were refined from those in our previous studies (Yamasue et al., 2004). As described in detail below, to accurately measure the volume of these structures in the present study, we developed an additional protocol for delineating the anterior boundary of the amygdala and the boundary between the amygdala and the hippocampus similar to those described in the previous literature (van Erp et al., 2002; Schumann et al., 2004). The dentate gyrus, CA fields, subiculum, presubiculum, and parasubiculum were referred to as the hippocampus, while the fornix, fimbria, and alveus were not included in the volumetric measurements. Tracing of the hippocampus was mainly performed in the sagittal plane; however, each slice was also edited in the axial and coronal planes to delineate the boundary of the ROIs as precisely as possible. Once drawn, hippocampal ROIs could be viewed in any plane and as a three-dimensional object for any further editing. Tracing began on the slice in the most lateral extent of the ventricular temporal horn on which the hippocampus was first visible. The outline represents the inferior border (determined by drawing a line through the white matter separating the hippocampus from the parahippocampal and fusiform gyri), superior border (determined by drawing a line through the alveus separating the hippocampus from the lateral ventricles), and the posterior border (determined by following the structure to its furthest posterior extent). More medially, the anterior hippocampus was separated from the amygdala by a thin line of white matter between the two structures (alveus). Tracing of amygdala boundaries began by defining the borders in coronal sections starting with the most caudal level in which the amygdala was visible. At its caudal extent, the amygdala is bordered dorsally by the substantia innominata, laterally by the putamen, and ventrally by the temporal horn of the lateral ventricle. The medial

surface of the amygdala abuts the optic tract. Proceeding rostrally, it is bordered dorsally by fibers of the anterior commissure as well as the substantia innominata. The lateral border is formed by white matter of the temporal lobe. The ventral surface is formed by the temporal horn of the lateral ventricle. However, because the hippocampus often appears to be fused with the ventral surface of the amygdala, a more reliable boundary is the alveus, the white matter that forms the dorsal surface of the hippocampus. In more rostral sections, the hippocampus decreases in size and the entorhinal cortex begins to form part of the medial surface of the amygdala. At this point, a thin band of white matter separates the amygdala from the entorhinal cortex. In most rostral sections, the dorsomedial surface of the amygdala forms a portion of the medial surface of the brain. The amygdala is bordered laterally by white matter of the temporal lobe, ventrally by the temporal horn of the lateral ventricle and by subamygdaloid white matter, and ventromedially by the entorhinal cortex. At the rostral pole of the amygdala, the outlining rules are very similar to what has just been described above. However, the gray-matter/white-matter boundaries are more difficult to delineate. Therefore, it was necessary to confirm the rostral boundary of the amygdala by reviewing the outlines in sagittal images. For interrater reliability, two raters (T.O. and M.A.R.) blind to group membership independently traced ROIs. The raters traced ROIs on every slice in the whole sample of 25 subjects. The intraclass correlation coefficient was 0.94/0.98 for left/right amygdala, and 0.96/0.96 for left/ right hippocampus, respectively. Intrarater reliability, computed by using all of the slices from one randomly selected brain and measured by one rater (T.O.) at two separate times (approximately 1 month apart), was N0.95 for all structures. Total gray matter, white matter, and cerebrospinal fluid volumes were calculated from the voxel based morphometry (VBM) procedure using SPM2 (Good et al., 2001). Intracranial content (ICC) was then calculated by summing up total gray matter, white matter, and cerebrospinal fluid volume. To validate this method, the ICCs of an independent sample of 50 adult subjects were measured by both the VBM and intensity-based semiautomated segmentation procedure using ANALYZE PC 3.0 (Yamasue et al., 2004). We then confirmed that the calculated intraclass correlation coefficient (ICC) was acceptable (0.96). 2.4. Image processing for VBM Image analysis using SPM 2 software (Wellcome Department of Cognitive Neurology, Institute of Neurology, London, UK) running in MATLAB 6.5 (Mathworks, Sherborn, MA) was the same as in our previous study (Yamasue et al., 2003) in order to test the relationship between the present and previous findings. Briefly, images were first spatially normalized into the standard space of Talairach and Tournoux (1988). Normalized images were then segmented into the gray matter, white matter, cerebrospinal fluid, and skull/scalp compartments using an automated and operator-independent process. The segmentation step incorporates an image density non-uniformity correction to address image density variations caused by different positions of cranial structures within the MRI head coil. The spatially normalized segments of gray and white matter were smoothed with a 12-mm full-width at half-maximum isotropic Gaussian kernel to accommodate individual variability in the sulcal and gyral anatomy. 2.5. Statistical analysis The effect of PTSD diagnosis on manually traced volumes was assessed using repeated measures analysis of variance (ANOVA) adopting relative volumes as the dependent variable, diagnosis as the between-subject factor, and region (amygdala/hippocampus) and hemisphere (left/right) as the within-subject factors. Individual difference in ICC is considered to be one of the major contributors to

3. Results 3.1. PTSD-history versus no PTSD-history: ROI volumes Because the Shapiro-Wilks test identified no significant deviation from normalcy (left amygdala: PTSD-history group: P =0.317; no PTSDhistory group: P= 0.133; right amygdala: PTSD-history group: P= 0.755; no PTSD-history group: P = 0.923; left hippocampus: PTSD-history group: P= 0.199; no PTSD-history group: P =0.618; right hippocampus: PTSD-history group: P = 0.467; no PTSD-history group: P = 0.674), absolute volumes of ROIs were analyzed by two-way ANOVA with the between- subject factor of Group (PTSD vs. No PTSD) and the withinsubject factors of hemisphere (left vs. right) and ROI (amygdala and hippocampus) (Table 2). Total intracranial volume was set as a controlling

The degrees of freedom were [1,23] for all statistics.

0.03 0.02 0.17 0.08

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individual differences in regional brain volume. Therefore, the relative volumes [100 × absolute ROI volume]/(ICC)] were used as the dependent variable. Of note, the statistical conclusions reported below remained the same when ANCOVA with absolute volume as the dependent variable and ICC as the covariate were used. The level of statistical significance was set at P b 0.05. Statistical analyses were performed using an analysis of covariance model (Friston et al., 1990). To account for global anatomical variations, the intracranial volume calculated from the VBM procedure was treated as a confounding covariate. To detect the neuroanatomical correlates of medial temporal volumetric abnormality, statistical analysis treated intracranial volume as a confounding covariate, and the volumetric measures showing a significant PTSD effect as the covariate of interest. The resulting set of voxel values for each contrast constituted a statistical parametric map of the t-statistic (SPM{t}). The SPM{t}s were displayed at an uncorrected threshold of P b 0.001 for graphical reporting. Family Wise Error-corrected P b 0.05 was conservatively employed to detect findings within the searched volumes (SV) based on our previous study (Yamasue et al., 2003). Furthermore, the diagnosis difference in the significant correlation between the medial temporal volumetric measure and the reduced gray matter density in the ACC was tested using a condition by covariates interaction analysis. This interaction analysis treated diagnosis as a condition, the volumetric measure as covariate of interest, and intracranial volume as confounding covariate. The threshold for statistical significance was set at P b 0.05, since the interaction analysis was post-hoc at a single voxel showing a significant correlation.

§

0.58 0.56 0.05 4.51 N.001 320.02 0.63 0.24 0.03 0.02 0.18 0.09 0.03 0.02 0.16 0.09 0.03 0.02

0.38 0.31 2.67 1.33 0.35 0.25 2.34 1.36 0.38 0.31 2.67 1.13 0.35 0.25 2.56 1.14

⁎Calculated by the following formula: absolute volume/intracranial content (ICC) ⁎ 100. ICC was not different between groups (PTSD subjects, mean, 1537 ml [S.D. = 114]; non-PTSD subjects, mean, 1521 ml [S.D. = 146]; t[23] = 0.29, p = 0.774).

2.11

0.05

0.05

F .628 P .241 Mean

S.D.

Mean

S.D.

Mean

S.D.

Mean

S.D.

F

Absolute volume (ml) Hippocampus Amygdala Relative volume (%)* Hippocampus Amygdala

Variables

0.18 0.07

t

2.1 0.44

P t

0.79 0.02

P F

9.85

P

Group × ROI ROI

6.69

Group × hippocampus Group Left hemisphere Left hemisphere

Right hemisphere Victims without PTSD (n = 16) Victims with PTSD (n = 9)

Right hemisphere Table 2 Volumetric measures.

0.01

t-test Repeated measures analysis of variance§

Group × amygdala

P

M.A. Rogers et al. / Psychiatry Research: Neuroimaging 174 (2009) 210–216

Fig. 1. Bilateral volume (expressed as a percentage of total intra-cranial volume) of the hippocampus and amygdala of the PTSD-history and No PTSD-history groups).

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M.A. Rogers et al. / Psychiatry Research: Neuroimaging 174 (2009) 210–216

variable. There was a significant interaction of Group and ROI such that the trauma victims with PTSD-history (M= 1.132, S.D.=0.243) had smaller amygdala volumes (ml) than did those without a diagnosis of PTSD (M =1. 345, S.D.=0.244); F(1,23) =7.095, P= 0.014. Independent samples t-tests confirmed that, while the amygdala volumes differed significantly between the two groups, hippocampal volume did not differ (Fig. 1). An identical analysis but with absolute volumes and with total intracranial volume as a control variable did not alter the statistical outcome (Fig. 1). 3.2. Correlation between left amygdala volume and PTSD symptoms Spearman correlations were conducted to test for an association between relative amygdala volume in the PTSD-history group and severity (sum of symptom frequency and symptom intensity) as indexed by the CAPS. There was a significant negative correlation for the avoidance subscale, r(9) = −0.752, P = 0.019. Spearman correlations also confirmed no significant correlations between right amygdala volume and PTSD symptoms in the PTSD-history group, as well as no significant correlations between either left or right amygdala volume and PTSD symptoms in the no PTSD-history group. 3.3. Correlation between left amygdala volume and left anterior cingulate gray matter density The positive correlation between left amygdala volume and the gray matter density in anterior cingulate cortex (FWE-corrected

Fig. 2. MRI indicating the position in the left anterior cingulate cortex where adjusted VBM responses correlate with left amygdala volume in the PTSD-history group, but not the no PTDS-history group (refer to scatterplot). The data for the left anterior cingulate were reported inYamasue et al. (2003).

P = 0.013, [−6 16 34], z = 3.54) as well as that in left amygdala (P b 0.001, z = 3.88, [−18 12 − 24]) was significant in the subjects with a history of PTSD but not in those without PTSD history (P = 0.43, z = 0.18). Interaction analysis showed a significant difference in the correlation between diagnostic groups in ACC (z = 2.71, P = 0.003) and in amygdala (z = 3.51, P b 0.001). These results indicate that the reduced left amygdala volume showed a significant positive correlation with the reduced gray matter density of the ACC and amygdala specifically in those subjects with a history of PTSD (Yamasue et al., 2003) (Fig. 2). 4. Discussion The purpose of this study was to determine if there is evidence of structural deficits associated with PTSD consistent with the theory that PTSD is due to a failure of normal fear-extinction. According to this failed fear-extinction hypothesis, abnormality of both the ACC and the amygdala may be expected to be associated with PTSD. Accordingly, as the PTSD-history participants in the present study have previously been shown to have reduced left hemisphere ACC gray matter density relative to the no PTSD-history group (Yamasue et al., 2003) as well as altered integrity of adjacent white matter (Abe et al., 2006), it was predicted that there would be reduced volume of the left amygdala in the PTSDhistory group relative to the no PTSD-history group. The findings were as predicted. The present results demonstrated bilaterally smaller amygdala volume in the patients with a history of PTSD compared with those without a history of PTSD. Smaller left amygdala volume was further associated with greater severity of PTSD avoidance symptoms and with reduced gray matter density in the left ACC only in the PTSD-history group. That this has not been observed previously is perhaps not surprising. Firstly, relatively few studies have looked at amygdala volume, and those that have done so employed tracing methods that are inadequate to accurately delineate the anterior boundary of the amygdala and distinguish the amygdala from the hippocampus. Furthermore, previous studies have found that while chronic stress or repeated exposure to stress may be associated with hypertrophy of amygdala neurons, acute stress is not (Vyas et al., 2003; Mitra et al., 2005). This may account for differences in findings between studies of persons such as combat veterans (who have been exposed repeatedly to stress) and who show no reduction in amygdala volume and the present study, where exposure to a single acute stressor is associated with reduced amygdala volume. The present finding is consistent with studies that have reported amygdala volume reduction in patients with fear- and anxiety-related disorders (Massana et al., 2003; Milham et al., 2005). One previous study reported amygdala volume reduction in individuals with intrusive recollection, a partial symptom of PTSD (Matsuoka et al., 2003). A recent study further showed a nonlinear association between the total lifetime number of traumatic episodes experienced and amygdala gray matter volume in healthy adults who had experienced the World Trade Center disaster on 9/11/01 (Ganzel et al., 2008). The present examination also revealed an inter-relationship between smaller amygdala volume and reduced gray matter density of the ACC in the left hemisphere specific to those participants with a history of PTSD. In addition, it is possible that abnormality of both of the main structures participating in fear extinction might contribute to lasting stress reactions observed in PTSD. Previous animal studies have shown that successful extinction of fear reaction requires interaction between synaptic plasticity of the amygdala and the ACC (Milad and Quirk, 2002; Herry and Mons, 2004). A recent functional MRI study further extended the interplay between the amygdala and the ACC in fear extinction to humans (Phelps et al., 2004). Therefore, the present study suggests the possibility that some pathological process associated with PTSD may facilitate a positive structural relationship between smaller size of the amygdala and smaller size of the ACC. However, further work is necessary to assess this possibility.

M.A. Rogers et al. / Psychiatry Research: Neuroimaging 174 (2009) 210–216

In addition to the differences in amygdala volume, there is the observation that amygdala volume was significantly associated with PTSD symptomatology as indexed by the avoidance measure of the CAPS in the left hemisphere but not the right hemisphere. Furthermore, left ACC gray matter density as reported in Yamasue et al. (2003) was significantly positively correlated with left amygdala volume reduction only in the PTSD-history group. This gives further support to the hypothesis that PTSD may involve a failure of normal extinction of learned fear secondary to dysfunction of the normal concerted action of the ACC and the amygdala. The present finding that bilateral hippocampus volume did not differ significantly between the two groups is consistent with the results of our VBM analysis (Yamasue et al., 2003). Although some previous studies have reported reduced hippocampal volume in subjects with PTSD (reviewed in Rauch et al., 2006; Letizia et al., 2007), several studies have reported no such difference (Bonne et al., 2001; Schuff et al., 2001; Yamasue et al., 2003). A recent meta-analysis (Woon and Hedges, 2008) suggested that reduced hippocampus volume may be associated with greater severity of PTSD. The present study further supports the notion that small hippocampus may be associated with chronic and/or severe nature of illness with psychiatric comorbidities, while a few studies have found small hippocampus in subjects with recent onset PTSD (Lindauer et al., 2004; Wignall et al., 2004). There are some potential limitations to the present study that must be discussed. Firstly, the sample size is somewhat small and this makes some of the statistical conclusions uncertain. In particular, while the amygdala was smaller bilaterally in the PTSD-history group, the finding that left, but not right, hemisphere amygdala volume was aassociated with PTSD symptomatology suggests that right hemisphere volume was not directly associated with PTSD diagnosis per se. Secondly, only one member of the PTSD-history group had the condition at the time of scanning. Thus it is uncertain to what extent the amygdala volume reduction identified here is present during the illness. It may be that volumes had recovered somewhat after remission, or conversely, that reduction may be associated with longer-term changes that persist after remission. A further point that must be considered is that a previous whole-brain VBM study of this same sample (Yamasue et al., 2003) did not identify any significant difference in bilateral amygdala volume. This raises the question as to which analysis is correct. In this respect it is worth noting that VBM may be insensitive to small and localized reductions in gray matter volume due to distortions caused by spatial standardization of images prior to analysis (Wright et al., 1999). Thus, it is quite feasible that the prior VBM analysis failed to detect the present reduction in amygdala volume. A further point that must be considered is that the location of dorsal ACC in the current study is different from those of rostral and ventral ACC identified in recent human fear conditioning studies (Phelps et al., 2004, Milad et al., 2005, Kalisch et al., 2006, Milad et al., 2007a). In addition, Milad et al. (2007b) found that increased thickness of a region of the ACC similar to that of interest in the present study to be correlated with increased rather than decreased fear. Therefore, the current results should be carefully interpreted in the context of fear extinction. However, Vogt et al. (2005) noted that a region of the dorsal ACC very close to that identified in the present study is associated with the modulation of fear. In conclusion, the current manually traced volumetry demonstrated bilaterally smaller amygdala in acute trauma victims with a history of PTSD compared to those with no history of PTSD. Furthermore, reduced left amygdala volume showed a significant negative correlation with greater severity of PTSD avoidance symptoms and with reduced left ACC gray matter density in the PTSDhistory group. Thus, the present findings provide in vivo brain morphological evidence consistent with the failure-of-fear-extinction theory of PTSD.

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