**Abnormal Brain Density in Victims of Rape with PTSD in Mainland China: A Voxel-Based Analysis of Magnetic Resonance Imaging Study**

Shuang Ge Sui1, Ling Jiang Li1, Yan Zhang1, Ming Xiang Wu2 and Mark E. King3 *1Mental Health Institute, Second Xiangya Hospital, Central-South University 2ShenZhen People's Hospital 3Faculty of Education, The University of Hong Kong, HHSAR P. R. China* 

### **1. Introduction**

374 Neuroimaging for Clinicians – Combining Research and Practice

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Posttraumatic stress disorder (PTSD) is a relatively common and predictable psychological syndrome (Miller, 1999). PTSD occurs in a proportion of individuals exposed to severe psychological trauma (Kasai et al., 2006) and in which the individual responds with fear, helplessness, or horror (Danckwerts & Leathem, 2003). Individuals with PTSD suffer from intrusive memories about the traumatic event, persistent avoidance of stimuli associated with the trauma, and persistent symptoms of increased arousal. These symptoms become uncontrollable and disabling (Bremner & Charney, 1994) that serious threaten human health and social function. Due to its debilitating nature, PTSD has emerged as an important public health problem in the general population (Sareen et al., 2007).

In recent years, a great deal of research has been directed towards understanding the etiology, phenomenology, neurobiology, clinical characteristics and treatment of PTSD (Nemeroff et al., 2006). However, a number of core psychological processes underlying PTSD have yet to be elucidated (Shin et al., 2006; Liberzon & Sripada, 2008). Over the past decade, findings from neuroimaging studies have allowed for tremendous advances in our understanding of the experience of emotions in healthy individuals and the dysregulation of these processes associated with PTSD. These studies have been useful in both generating hypotheses on the neurobiology of normative human responses to trauma and complementing our understanding of the wide-ranging alterations in trauma survivors who develop PTSD.

Structural neuroimaging studies have focused primarily on hippocampal volumetry (Geuze et al., 2005) as well as the prefrontal cortex (Geuze et al., 2008a) and other brain structures. Hippocampal morphology has been correlated with severity of PTSD symptomatology (Gilbertson et al., 2002; Villarreal & King, 2004). However, the results have been inconsistent, with studies reporting significant reductions or increases, as well as unchanged volumes. For example, studies have shown that patients with PTSD are associated with bilateral lower hippocampal volume (Bossini & Castrogiovanni, 2007; Bremner et al., 2003; Emdad et al., 2006; Lindauer et al., 2004a; Vythilingam et al., 2005; Li et al., 2006), which are

Abnormal Brain Density in Victims of Rape with PTSD in Mainland

in cerebellum sub-areas.

et al., 2008; Luders et al., 2004).

China: A Voxel-Based Analysis of Magnetic Resonance Imaging Study 377

left, right, and total cerebellum were smaller in maltreated children and adolescents with PTSD. They also found that cerebellar volume was positively correlated with the age of onset of the trauma that led to PTSD and negatively correlated with the duration of the trauma (Bellis & Kuchibhatla, 2006). Even with these findings, no precise results were found

In addition to findings related to the hippocampus and medial prefrontal cortex, many current functional neuroimaging studies have identified that other brain areas such as the prefrontal cortex, temporal lobe, parietal lobe, limbic lobe and cerebellum may also be implicated in PTSD (Bonne et al., 2003; Lanius et al., 2005; Molina et al., 2010; Bremner, 2006, Bremner et al., 2008; Geuze et al., 2008b). Brain functional imaging studies from patients with PTSD showing increased amygdala function and decreased medial prefrontal/anterior cingulate function during fear acquisition are hypothesized to represent a neural correlate of the failure of extinction seen in PTSD (Bremner , 2006, Bremner et al., 2008). A functional magnetic resonance imaging study comparing veterans with and without PTSD reveals that those with PTSD had overactivation of the temporal gyrus during the encoding phase, and underactivation of the bilateral middle temporal gyrus in the retrieval phase (Geuze et al., 2008b). Another functional magnetic resonance imaging study comparing PTSD patients and control subjects' connectivity maps in the left ventrolateral thalamus reveals that PTSD subjects had higher covariations between activations in he left ventrolateral thalamus and in the right insula, left parietal lobe, right middle frontal gyrus, and superior temporal gyrus (Lanius et al., 2005). Fluorodeoxyglucose positron emission tomography (FDG-PET) researches also indicate relatively diminished activity in the limbic, frontal and prefrontal cortex; relatively augmented activity in the fusiform cortex and in the cerebellum in patients with PTSD (Bonne et al., 2003; Molina et al., 2010). The findings of functional neuroimaging studies suggest that there are more brain areas that may be affected in PTSD, but only a few studies have found corresponding structural abnormalities in these brain areas. In contrast to the considerable research on subcortical structure volumetry, few studies to date have been directed to gray matter reductions in the cortex. It is evident that structural neuroimaging studies will allow for the testing of hypotheses of an association between PTSD and abnormal gray matter. Although volumetry findings reveal changes in the volume of specific brain regions, most of these studies defined particular regions-of-interests (ROIs) and measured their size and hemispheric asymmetry using traditional morphometric techniques with high-resolution magnetic resonance images (MRI). The disadvantage of this method is that some important brain areas may be neglected, and the process of drawing ROIs may introduce additional errors. Furthermore, the measurement of volume may not accurately reflect changes in the internal structure of the brain. In recent years, a fully automated voxel-based morphometry (VBM) technique has allowed for the examination of cerebral asymmetries across the entire brain directly (Corbo et al., 2005; Kasai et al., 2008; Luders et al., 2004), which can compensate for the subjectivity of ROI approaches. The VBM technique has been used for assessing regional gray matter density (GMD) (per unit volume in native space) in PTSD patients and revealed abnormal GMD in the hippocampus, anterior cingulate cortex, and insula (Emdad et al., 2006; Jatzko et al., 2006a; Richert et al., 2006; Kasai

Most previous PTSD studies in the West focused on the disorder caused by various traumatic events, such as war (Vythilingam et al., 2005; Bremner et al., 1995; Pavic et al., 2007), disaster (Jatzko et al., 2006a, b; Abe et al., 2006; Li et al., 2006; Yamasue et al., 2003), and sexual abuse (Bremner et al., 2003; Tupler&De Bellis, 2006). Although considerable research has focused on rape-related PTSD, limited studies have been carried out in the

considered to be either due to atrophy of the hippocampus as a consequence of suffering from PTSD due to excessive stress (Bremner et al., 1995; Gurvits et al., 1996) or that hippocampal volume to be a risk factor for developing PTSD (Gilbertson et al., 2002). Other studies report unchanged hippocampal volumes in female patients with chronic PTSD traumatized by intimate partner violence (Fennema-Notestine et al., 2002), those traumatized by witnessing a plane crash at the same air show (Jatzko et al., 2006a), elderly PTSD patients (Golier et al., 2006), and adult burn patients (Winter & Irle, 2004). Opposite trends in abused juveniles were found in other studies. (Tupler & De Bellis, 2006). A recent meta-analysis, however, confirmed the presence of significantly smaller hippocampal and left amygdala volumes in patients with PTSD compared to controls with and without trauma exposure (Karl et al., 2006). The findings of previous studies suggest that abnormal hippocampal volume was not a necessary and sufficient condition of PTSD.

Several studies have shown that the medial prefrontal cortex, which includes the anterior cingulate cortex and medial frontal cortex, are involved in the process of extinction of fear conditioning and the retention of extinction (Milad et al., 2006). Research on abnormalities in the prefrontal cortex in PTSD patients suggested decreased volume (Fennema-Notestine et al., 2002; Carrion et al., 2001; Richert et al., 2006; Hakamata et al., 2007), while some findings suggested increased volume of the middle-inferior and ventral regions of the prefrontal cortex (Richert et al., 2006).

The cerebellum has been considered only as a classical subcortical center for motor control (Botez, 1993). Botez et al. found that the patients with bilateral cerebellar damage showed deficits in the non-motor and behavioural functions including execution, attention, learning, and cognition (Botez, 1993; Ciesielski & Knight, 1994; Gao et al., 1996). Gao et al. found that the lateral cerebellar output (dentate) nucleus is not activated by the control of movement per se, but is strongly engaged during passive and active sensory tasks (Gao et al., 1996). Recent research of the cerebellum's contribution to cognitive processing and emotional processing have increased enormously, showing that the cerebellum is responsible for sensory perception, learning, memory, attention, linguistic, emotional control and conflict resolution processing (Mandolesi et al., 2001; Bischoff-Grethe et al., 2002; Vokaer et al., 2002; Claeys et al., 2003; Guenther et al., 2005; Allen et al., 2005; Konarski et al., 2005; Schmahmann & Caplan, 2006; Gianaros et al., 2007; Schweizer et al., 2007).

Anatomical studies revealed that via the thalamus, the cerebellum interacts with multiple areas of the prefrontal cortex and subcortex limbic lobe (Middleton & Strick, 2001; Zhu et al., 2006). The cerebellum influences several areas of the prefrontal cortex via the thalamus (Middleton & Strick, 2001). Gold and Buckner found a region in the right lateral cerebellum which exhibited a pattern similar to the left inferior frontal gyrus during semantic decisions on words and phonological decisions on pseudowords (Gold & Buckner, 2002). Patients with degenerative cerebellar diseases show high rates of cognitive impairment or psychiatric symptoms (Leroi et al., 2002; Liszewski et al., 2004), and neuroimaging studies have found that mood disorders were activated in the cerebellum (Liotti et al., 2000; Phan et al., 2002). The study by Gianaros et al. found that healthy individuals show heightened stressor-induced neural activation in the cingulate cortex, bilateral prefrontal cortex, and cerebellum while performing a standardized Stroop color-word interference task (Gianaros et al., 2007); however, studies of the cerebellum in PTSD patients have been very limited. In two positron emission tomography (PET) studies, abnormal activities in the cerebellum of PTSD subjects were found, including higher regional cerebral blood flow (Bonne et al., 2003) and augmented glucose absorption activity (Molina et al., 2010). Bellis et al. found that the

considered to be either due to atrophy of the hippocampus as a consequence of suffering from PTSD due to excessive stress (Bremner et al., 1995; Gurvits et al., 1996) or that hippocampal volume to be a risk factor for developing PTSD (Gilbertson et al., 2002). Other studies report unchanged hippocampal volumes in female patients with chronic PTSD traumatized by intimate partner violence (Fennema-Notestine et al., 2002), those traumatized by witnessing a plane crash at the same air show (Jatzko et al., 2006a), elderly PTSD patients (Golier et al., 2006), and adult burn patients (Winter & Irle, 2004). Opposite trends in abused juveniles were found in other studies. (Tupler & De Bellis, 2006). A recent meta-analysis, however, confirmed the presence of significantly smaller hippocampal and left amygdala volumes in patients with PTSD compared to controls with and without trauma exposure (Karl et al., 2006). The findings of previous studies suggest that abnormal

Several studies have shown that the medial prefrontal cortex, which includes the anterior cingulate cortex and medial frontal cortex, are involved in the process of extinction of fear conditioning and the retention of extinction (Milad et al., 2006). Research on abnormalities in the prefrontal cortex in PTSD patients suggested decreased volume (Fennema-Notestine et al., 2002; Carrion et al., 2001; Richert et al., 2006; Hakamata et al., 2007), while some findings suggested increased volume of the middle-inferior and ventral regions of the prefrontal

The cerebellum has been considered only as a classical subcortical center for motor control (Botez, 1993). Botez et al. found that the patients with bilateral cerebellar damage showed deficits in the non-motor and behavioural functions including execution, attention, learning, and cognition (Botez, 1993; Ciesielski & Knight, 1994; Gao et al., 1996). Gao et al. found that the lateral cerebellar output (dentate) nucleus is not activated by the control of movement per se, but is strongly engaged during passive and active sensory tasks (Gao et al., 1996). Recent research of the cerebellum's contribution to cognitive processing and emotional processing have increased enormously, showing that the cerebellum is responsible for sensory perception, learning, memory, attention, linguistic, emotional control and conflict resolution processing (Mandolesi et al., 2001; Bischoff-Grethe et al., 2002; Vokaer et al., 2002; Claeys et al., 2003; Guenther et al., 2005; Allen et al., 2005; Konarski et al., 2005;

Anatomical studies revealed that via the thalamus, the cerebellum interacts with multiple areas of the prefrontal cortex and subcortex limbic lobe (Middleton & Strick, 2001; Zhu et al., 2006). The cerebellum influences several areas of the prefrontal cortex via the thalamus (Middleton & Strick, 2001). Gold and Buckner found a region in the right lateral cerebellum which exhibited a pattern similar to the left inferior frontal gyrus during semantic decisions on words and phonological decisions on pseudowords (Gold & Buckner, 2002). Patients with degenerative cerebellar diseases show high rates of cognitive impairment or psychiatric symptoms (Leroi et al., 2002; Liszewski et al., 2004), and neuroimaging studies have found that mood disorders were activated in the cerebellum (Liotti et al., 2000; Phan et al., 2002). The study by Gianaros et al. found that healthy individuals show heightened stressor-induced neural activation in the cingulate cortex, bilateral prefrontal cortex, and cerebellum while performing a standardized Stroop color-word interference task (Gianaros et al., 2007); however, studies of the cerebellum in PTSD patients have been very limited. In two positron emission tomography (PET) studies, abnormal activities in the cerebellum of PTSD subjects were found, including higher regional cerebral blood flow (Bonne et al., 2003) and augmented glucose absorption activity (Molina et al., 2010). Bellis et al. found that the

hippocampal volume was not a necessary and sufficient condition of PTSD.

Schmahmann & Caplan, 2006; Gianaros et al., 2007; Schweizer et al., 2007).

cortex (Richert et al., 2006).

left, right, and total cerebellum were smaller in maltreated children and adolescents with PTSD. They also found that cerebellar volume was positively correlated with the age of onset of the trauma that led to PTSD and negatively correlated with the duration of the trauma (Bellis & Kuchibhatla, 2006). Even with these findings, no precise results were found in cerebellum sub-areas.

In addition to findings related to the hippocampus and medial prefrontal cortex, many current functional neuroimaging studies have identified that other brain areas such as the prefrontal cortex, temporal lobe, parietal lobe, limbic lobe and cerebellum may also be implicated in PTSD (Bonne et al., 2003; Lanius et al., 2005; Molina et al., 2010; Bremner, 2006, Bremner et al., 2008; Geuze et al., 2008b). Brain functional imaging studies from patients with PTSD showing increased amygdala function and decreased medial prefrontal/anterior cingulate function during fear acquisition are hypothesized to represent a neural correlate of the failure of extinction seen in PTSD (Bremner , 2006, Bremner et al., 2008). A functional magnetic resonance imaging study comparing veterans with and without PTSD reveals that those with PTSD had overactivation of the temporal gyrus during the encoding phase, and underactivation of the bilateral middle temporal gyrus in the retrieval phase (Geuze et al., 2008b). Another functional magnetic resonance imaging study comparing PTSD patients and control subjects' connectivity maps in the left ventrolateral thalamus reveals that PTSD subjects had higher covariations between activations in he left ventrolateral thalamus and in the right insula, left parietal lobe, right middle frontal gyrus, and superior temporal gyrus (Lanius et al., 2005). Fluorodeoxyglucose positron emission tomography (FDG-PET) researches also indicate relatively diminished activity in the limbic, frontal and prefrontal cortex; relatively augmented activity in the fusiform cortex and in the cerebellum in patients with PTSD (Bonne et al., 2003; Molina et al., 2010). The findings of functional neuroimaging studies suggest that there are more brain areas that may be affected in PTSD, but only a few studies have found corresponding structural abnormalities in these brain areas. In contrast to the considerable research on subcortical structure volumetry, few studies to date have been directed to gray matter reductions in the cortex. It is evident that structural neuroimaging studies will allow for the testing of hypotheses of an association between PTSD and abnormal gray matter. Although volumetry findings reveal changes in the volume of specific brain regions, most of these studies defined particular regions-of-interests (ROIs) and measured their size and hemispheric asymmetry using traditional morphometric techniques with high-resolution magnetic resonance images (MRI). The disadvantage of this method is that some important brain areas may be neglected, and the process of drawing ROIs may introduce additional errors. Furthermore, the measurement of volume may not accurately reflect changes in the internal structure of the brain. In recent years, a fully automated voxel-based morphometry (VBM) technique has allowed for the examination of cerebral asymmetries across the entire brain directly (Corbo et al., 2005; Kasai et al., 2008; Luders et al., 2004), which can compensate for the subjectivity of ROI approaches. The VBM technique has been used for assessing regional gray matter density (GMD) (per unit volume in native space) in PTSD patients and revealed abnormal GMD in the hippocampus, anterior cingulate cortex, and insula (Emdad et al., 2006; Jatzko et al., 2006a; Richert et al., 2006; Kasai et al., 2008; Luders et al., 2004).

Most previous PTSD studies in the West focused on the disorder caused by various traumatic events, such as war (Vythilingam et al., 2005; Bremner et al., 1995; Pavic et al., 2007), disaster (Jatzko et al., 2006a, b; Abe et al., 2006; Li et al., 2006; Yamasue et al., 2003), and sexual abuse (Bremner et al., 2003; Tupler&De Bellis, 2006). Although considerable research has focused on rape-related PTSD, limited studies have been carried out in the

Abnormal Brain Density in Victims of Rape with PTSD in Mainland

images were performed in the general linear model.

rather than corrections for the whole brain.

**3. Results** 

diagnosis of PTSD.

**2.2 MRI data acquisition** 

**2.3 MRI data analysis** 

China: A Voxel-Based Analysis of Magnetic Resonance Imaging Study 379

(Ruggiero et al., 2003). In addition, two independent, clinically experienced psychiatrists interviewed VoR subjects using the Clinician-Administered PTSD Scale (CAPS) (Blake et al., 1995). The PCL-C was used to predict PTSD diagnoses, and the CAPS was used to differentiate PTSD and non-PTSD VoR subgroups. A senior psychiatrist confirmed the final

Images were obtained from using a research-dedicated Siemens Avanto 1.5 Tesla MRI scanner. The T1-weighted anatomical images were acquired using a three-dimensional gradient-echo sequence, with TR=11 msec, TE=4.94 msec, number of averages=1, matrix=256×224 pixels, field of view=256mm×224mm, with a flip angle of 15°. 176 sagittal slices with a 1 mm slice thickness were acquired with no interslice gap. There was a voxel

Voxel-based morphometry was implemented by using the Statistical Parametric Mapping software (SPM2) (Wellcome Department of Imaging Neuroscience, London, England; www.fil.ion.ucl.ac.uk) (Friston et al., 1995). First, images were spatially normalized to the Montreal Neurological Institute (MNI) space with the standard T1-MRI template (Mazziotta et al., 1995) implemented in the SPM2 program, and re-sliced into a final voxel size of 1×1×1mm3 using tri-linear interpolation. The spatially normalized images were then segmented into three compartments: gray matter, white matter and cerebrospinal fluid. Furthermore, a Jacobian determinant was not introduced to modulate the resulting gray matter images so the voxel's values indicate the absolute density of the local gray matter. Finally, the segmented gray matter images from VoR with PTSD, VoR without PTSD, and HC were smoothed with a 12-mm full-width at half-maximum isotropic Gaussian kernel (Ashburner & Friston, 2000). The result of between-groups comparisons of gray matter

Because we are particularly interested in exploring increases/decreases in GMD in VoR with PTSD compared to VoR without PTSD and HC, two-sample t-tests were performed in the VBM analysis in a voxel-by-voxel manner. Consistent with previous studies (Liberzon et al., 2007; Hou et al., 2007), the significance threshold was set to p < 0.005 corrected for multiple comparisons with a minimal cluster size of >50 voxels. The significant regions were superimposed onto SPM2's standard T1-weighted brain images. Based on previous research (Milad et al., 2006; Carrion et al., 2001; Richert et al., 2006; Hakamata et al., 2007; Bonne et al., 2003; Lanius et al., 2005; Molina et al., 2010; Bremner et al., 2006, 2008; Geuze et al., 2008b), we hypothesized that compared with HC, VoR with PTSD would show gray matter abnormalities in the prefrontal, temporal, parietal and limbic regions. We used the small volume correction (SVC) tool in the SPM2 package with the specific purpose of restricting comparisons to specific voxels located in these regions. This approach permits the implementation of hypothesis-driven analyses with corrections for the pre-specified ROIs

Following the initial interview, among the 23 VoR subjects, 13 met the DSM-IV diagnostic criteria for current PTSD and 10 VoR did not meet the criteria for PTSD. Based on the study

resolution of 1×1×1mm 3. The total acquisition time was 5 minutes and 34 seconds.

context of Mainland China. In this study, rape was defined as an event that occurred without the victim's consent that involved the use or threat of force to penetrate the victim's vagina or anus by penis, tongue, fingers, or object, or the victim's mouth by penis (Tjaden & Thoennes, 2000). Interestingly, evidence indicates that the incidence rate of PTSD induced by rape is the highest among all kinds of trauma (Rothbaum et al., 1992).

In the current study, we employed VBM to explore differences in GMD between victims of rape (VoR) with and without PTSD, as well as in healthy comparison (HC) subjects. Based on findings from previous neuroimaging studies (Milad et al., 2006; Carrion et al., 2001; Richert et al., 2006; Hakamata et al., 2007; Bonne et al., 2003; Lanius et al., 2005; Molina et al., 2010; Bremner et al., 2006, 2008; Geuze et al., 2008b), we hypothesized that VoR with PTSD would show structural changes in extensive brain areas, including the prefrontal, temporal, parietal and limbic regions, compared to VOR without PTSD and to HC.
