Cortical thinning of the right anterior cingulate cortex in spider phobia: A magnetic resonance imaging and spectroscopy study

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Research Report

Cortical thinning of the right anterior cingulate cortex in spider phobia: A magnetic resonance imaging and spectroscopy study

Q1 Q3

I.M.P. Linaresa,n, A.P. Jackowskid, C.M.F. Trzesniaka, K.C. Arraisa, M.H.N. Chagasa, J.R. Satob, A.C. Santosc, J.E.C. Hallaka, A.W. Zuardia, A.E. Nardif, N.C. Coimbrae, J.A.S. Crippaa a

Department of Neuroscience and Behavior, Ribeirão Preto Medical School, University of São Paulo, National Institute for Translational Medicine (INCT, CNPq), Ribeirão Preto, São Paulo, Brazil b Center of Mathematics, Computation and Cognition, Universidade Federal do ABC, Santo André, Brazil c Department of Medical Clinic Department, Division of Radiology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil d Laboratory of Interdisciplinary Neurosciences (LINC), Department of Psychiatry, Federal University of São Paulo, São Paulo, Brazil e Laboratory of Neuroanatomy & Neuropsychobiology, Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil f Laboratory of Panic and Respiration, Institute of Psychiatry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

art i cle i nfo

ab st rac t

Article history: Accepted 26 May 2014

Q2 There a lack of consistent neuroimaging data on specific phobia (SP) and a need to assess volumetric and metabolic differences in structures implicated in this condition.

Keywords:

To investigate possible metabolic (via 1H MRS) and cortical thickness abnormalities in

Specific phobia

spider-phobic patients compared to healthy volunteers. Participants were recruited via

Neuroimaging

public advertisement and underwent clinical evaluations and MRI scans. The study started

Anxiety

in 2010 and the investigators involved were not blind in respect to patient groupings. The

Fear

study was conducted at the Ribeirão Preto Medical School University Hospital of the

ACC

University of São Paulo, Brazil. Patients with spider phobia (n ¼19) were matched to 17 healthy volunteers with respect to age, education and socio-economic status. The spider SP group fulfilled the diagnostic criteria for spider phobia according to the Structured Clinical Interview for DSM-IV. None of the participants had a history of neurological, psychiatric or other relevant organic diseases, use of prescribed psychotropic medication or substance abuse. All imaging and spectroscopy data were collected with a 3 T MRI scanner equipped with 25 mT gradient coils in 30-minute scans. The Freesurfer image analysis package and LC Model software were used to analyze data. The hypothesis being tested was formulated before the data collection (neural correlates of SP would include the amygdala, insula, anterior cingulate gyrus and others). The results indicated the absence of metabolic

n

Correspondence to: Hospital das Clínicas, 3rd floor, Av. Bandeirantes, 3900, 14048-900 Ribeirão Preto, SP, Brazil. Fax: þ55 16 36022544. E-mail address: [email protected] (I.M.P. Linares).

http://dx.doi.org/10.1016/j.brainres.2014.05.040 0006-8993/& 2014 Published by Elsevier B.V.

Please cite this article as: Linares, I.M.P., et al., Cortical thinning of the right anterior cingulate cortex in spider phobia: A magnetic resonance imaging and spectroscopy study. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.040

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alterations, but thinning of the right anterior cingulate cortex (ACC) in the SP group when compared to the healthy control group (mean cortical thickness7SD: SP¼ 2.1170.45 mm; HC ¼2.1670.42 mm; t (34) ¼ 3.19, p ¼ 0.001 [35.45, 71.00,  23.82]). In spectroscopy, the ratios between N-acetylaspartate and creatine and choline levels were measured. No significant effect or correlation was found between MRS metabolites and scores in the Spider Phobia Questionnaire and Beck Anxiety Inventory (p40.05). The ACC is known to be related to the cognitive processing of fear and anxiety and to be linked with the conditioning circuit. The MRS findings are preliminary and need more studies. The finding of reduced ACC thickness in SP is in agreement with evidence from previous functional neuroimaging studies and highlights the importance of this brain area in the pathophysiology of SP. & 2014 Published by Elsevier B.V.

1.

Introduction

Specific phobia (SP) is characterized by unreasonable, excessive, and persistent fear in the presence or anticipation of feared situations or objects. SP is considered to be one of the most prevalent anxiety disorders and is subdivided into five Q4 subtypes (animal, blood-injection-injury, situational, natural events, and others) (Kessler et al., 2005). The most common subtype of SP is the animal subtype, which has a prevalence of 12.1% in women and 3.3% in men (Fredrikson et al., 1996). The etiology of animal SP remains uncertain, but available evidence suggests that constitutional and environmental factors may contribute to its etiology (Fyer, 1998). Environmental factors are related to specific events and to the conditioning process. Fear conditioning is essential for danger detection; however, in some cases it may also influence the emergence of disorders related to anxiety and fear (Sehlmeyer et al., 2009). It is important to mention that the outcomes of conditioning processes are dependent on vulnerability factors including differences in life experiences, perceived controllability, direct or vicarious experience, type and intensity of experience, and information transmission (Mineka and Zinbarg, 2006). Constitutional factors refer to genetic components and ensuing neural alterations. Some studies have supported the notion that genetic risk factors play a role in the etiology of SP (Kendler et al., 2001). Moreover, there are hypotheses that animal SP can be related to abnormalities in the encephalic aversion system (EAS), although the specific neural networks of the EAS involved in the physiopathology of SP are not completely understood. Several original articles and some reviews (Shin and Liberzon, 2001) pointed to the involvement of the amygdaloid complex (Morris et al., 1988; Phelps, 2005), the dorsal hippocampus (Zelikowsky et al., 2012), the nucleus accumbens (Luckett et al., 2001), the ventromedial hypothalamus (Wilent et al., 1994; Biagioni and Coimbra, 2012), the periaqueductal gray matter (Carrive et al., 1997; Vianna and Brandao, 2003), the corpora quadrigemina (Castellan-Baldan et al., 2006; Coimbra et al., 2006; Eichenberger et al., 2002), the insular cortex (van Well et al., 2012) thalamic nuclei, and some prefrontal neocortical regions (Mobbs et al., 2007) in both unconditioned and conditioned fear-related behaviors studied in laboratory animal- and human-based models of

fear and panic. Therefore, patients with animal SP would be expected to have abnormalities in this network. To assess brain responses and better comprehend animal SP, most studies have used functional neuroimaging techniques (fMRI, PET, and SPECT) to measure the effects of exposure to phobia-related images (Linares et al., 2012). The results are not totally homogeneous; however, many studies that investigated spider phobia identified neural correlates of autonomic (amygdala) and direct evaluation (e.g. insula, anterior cingulate gyrus, and left dorsomedial prefrontal cortex) of threatening stimuli (Straube and Miltner, 2006; Schienle et al., 2005; Dilger et al., 2003). In spite of that, little is known about other neuroimaging techniques and their possible contributions to the understanding of the neurobiology of animal SP. Newer structural, metabolic and functional neuroimaging techniques might contribute to a better understanding of the etiology and pathophysiology of SP. Magnetic resonance spectroscopy (MRS) and structural MRI provide in vivo neuroanatomical information and neurochemical quantification and, in association with functional neuroimaging techniques, can help uncover the neural substrates of SP. To the best of our knowledge, no study has investigated possible metabolite changes using proton magnetic resonance spectroscopy (1H MRS) in SP, and only two studies have used structural measures of cortical thickness (Rauch et al., 2004; Rosso et al., 2010) in this population. The insufficiency of consistent data and the need to assess volumetric differences in structures implicated in SP make the continuation of complementary studies in the area an issue of paramount importance. Thus, the aim of the present study was to investigate possible cortical thickness and metabolic abnormalities in spider-phobic patients compared to healthy volunteers.

2.

Results

2.1.

Cortical thickness

Cortical thickness measurements indicated thinning of the right ACC in the group with spider phobia compared to the HC group (mean cortical thickness7SD: SP ¼2.1170.45 mm,

Please cite this article as: Linares, I.M.P., et al., Cortical thinning of the right anterior cingulate cortex in spider phobia: A magnetic resonance imaging and spectroscopy study. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.040

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HC¼ 2.1670.42 mm; t (34) ¼3.19, p ¼0.001 [  35.45, 71.00,  23.82]). There were no differences in the left hemisphere (mean cortical thickness7SD: SP¼ 2.1270.41 mm, HC¼2.137 0.41 mm; t (34) ¼1.84, p ¼ns [38.89, 63.88,  35.98]). Post-hoc inspection of whole brain measures did not show other loci with comparable increases in cortical thickness. A general linear model was used to demonstrate the main difference between the groups (cortical thickness differences between SP patients and controls), as shown in Fig. 2. Cortical thickness did not change in the HC group.

2.2.

1

H MRS

To evaluate the influence of metabolites on the neurobiology of spider phobia, participants underwent 1H MRS scans. Both variables (NAA/Cr and Cho/Cr ratios) were tested using the Kolmogorov–Smirnov (normality) and Levene (homogeneity of variance) tests. No differences in NAA/Cr and Cho/Cr ratios were found in the cingulate gyrus of phobic patients compared to the HC group (Table 2). Additionally, no significant correlation was found between MRS metabolites, the SPQ, and the BAI (P40.05).

3.

3.1.

3

1

H MRS

MRS is a neuroimaging technique that allows in vivo chemical examination of brain tissues and has recently been used to address some questions regarding the neurobiology of anxiety disorders (Trzesniak et al., 2008). However, no study to date has investigated possible metabolite alterations that may occur in SP (Linares et al., 2012). Phobia patients are subject to persistent high levels of anxiety as a result of the increased discharge of excitatory neurotransmitters and pharmacological and morphological neuroplasticity, resulting in increased neuronal synaptic contacts and reorganization in the number of axons within limbic and paralimbic structures that can be associated with alterations in NAA concentrations (Grachev and Apkarian, 2000). Since the cingulate cortex is known to encompass specialized subdivisions related to cognitive, emotional, and nociceptive responses (Vogt et al., 1992) and that it plays a role in the neurobiology of animal SP, we hypothesized that

Discussion

In the present study, we used MRS and structural MRI to examine brain abnormalities in patients with spider phobia and HC subjects. According to the MRS results, there were no significant metabolite abnormalities in the brain of phobic patients. Structural analysis provided evidence that cortical thickness in the ACC of patients with spider phobia was reduced in comparison to the HC group. No other areas of activation or deactivation were found. Additionally, no gender-related differences were found with respect to neuroimaging results.

Fig. 2 – Regions with increased cortical thickness in spider SP group versus HC. Mean cortical thickness7SD: SP¼ 2.1170.45 mm, HC¼2.1670.42 mm; t (34)¼ 3.19, and p¼ 0.001 ( 35.45, 71.00, 23.82).

Fig. 1 – Volume of interest (VOI) – cingulated cortex: axial and coronal slices. Please cite this article as: Linares, I.M.P., et al., Cortical thinning of the right anterior cingulate cortex in spider phobia: A magnetic resonance imaging and spectroscopy study. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.040

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3.2.

Table 1 – Group characteristics.

Gender (F/M) Age (years) Handedness Education (years) Socio-economic SPQ BAI

Spider SP (n ¼ 18)

HC (n ¼ 17)

P

17:1 30.9478.06 18R:1L 14.3372.74 20.1175.45 115.3878.18 11.2776.56

16:1 29.4778.66 15R:2L 15.4172.15 24.0577.57 22.0076.76 5.6477.64

1 0.57 0.52 0.207 0.085 0.001n 0.025n

Values are presented as means7SD. SP, specific phobia subjects; SPQ, spider phobia questionnaire; BAI, Beck Anxiety Inventory. n Significant at Po0.05. Independent t-tests were used to assess between n-group differences in quantitative variables.

Table 2 – Mean (standard deviation) and t-test values for the NAA/Cr and Cho/Cr ratios of spider SP patients and HC cingulate gyrus.

NAA/Cre Cho/Cre

Spider SP (N¼ 19)

HC (N¼ 17)

T-test P

1.1670.14 0.8170.28

1.1670.12 0.7770.22

0.95 0.67

Cho, choline; Cr, creatine; NAA, N-acetylaspartate.

there could be metabolic alterations in the cingulate cortex in the spider SP group. According to our results, however, there were no metabolic alterations in the spider SP group compared to the HC group. Previous reports in the literature using MRS to investigate other anxiety disorders suggest that the absence of alterations may be explained by classical confounding factors such as age or gender of the participants, and the nature, duration, and severity of the illness (Grachev and Apkarian, 2000). The sequence used for slice selection and excitation (point-resolved spectroscopy – PRESS) produces fewer artifactual spectra than the other commonly used sequence (STEAM); however, it provides data about the concentrations of fewer metabolites (Trzesniak et al., 2010). The absence of metabolic alterations in our region of interest could also be related to the application of a single voxel methodology. Several studies have used single voxel MRS because it is faster than multivoxel MRS, but larger voxel sizes result in lower spatial resolution and does not allow for the evaluation of multiple regions. Finally, another possible drawback of our approach is the area of analysis (cingulate cortex), which may have limited our ability to detect activation in other brain regions that may be relevant to our understanding of SP, e.g., the amygdaloid complex. Combined research methods can help to clarify the relationships among functional, structural, and biochemical mechanisms underlying the psychological manifestations of anxiety disorders (Miller and Keller, 2000). The increasing availability of MRS may have an impact on the number and relevance of studies on anxiety disorders.

Cortical thickness

As mentioned before, structural neuroimaging research in SP is still in its early stages. To date, only two previous studies assessed cortical thickness in groups with SP. One of these studies showed bilateral increases in the insular cortex, pregenual anterior cingulate gyrus, posterior cingulate gyrus, and left visual cortical area (Rauch et al., 2004). The other study evaluated the morphology of the insula using MRI, and found no differences in terms of insular volume or thickness (Rosso et al., 2010). Considering some limitations that may have affected the results of these studies, such as the sample size and analyses restricted to certain structures, the need for additional investigations on cortical thickness in SP seems justified. Cortical thickness is an indicator of the cytoarchitectural integrity of cortical regions (Makris et al., 2007); however, the meaning of decreased cortical thickness is etiologically and pathogenically imprecise. In other words, it cannot be determined whether thinning of the ACC reflects decrease in synaptic or glial density, volume or number, or changes in their arrangement patterns (Makris et al., 2007). However, the presence of specific structural abnormalities may suggest a potential dysfunction involving this specific area. The ACC is part of the limbic system (Bush et al., 2000) and is a central structure in the mediation of anxiety effects such as phobic avoidance (Straube et al., 2009). The ACC has two subdivisions that perform cognitive (dorsal) and emotional/ affective (rostral and ventral) functions (Vogt et al., 1992; Bush et al., 2000). Our findings are restricted to the rostral part of the ACC. This region is primarily involved in assessing the salience of emotional and motivational information and the regulation of emotional responses (Devinsky et al., 1995). The recruitment of the ACC during painful stimulation overlaps with the activation of this structure in emotion-related disorders, such as post-traumatic stress (Liberzon and Martis, 2006) and anxiety (Etkin and Wager, 2007), as shown by imaging studies with human subjects (Wiech and Tracey, 2009). These findings indicate the possible involvement of the ACC in affective pain processing (Berna et al., 2010). Functional studies report an association between the right ACC and fear conditioning (Dunsmoor et al., 2007). The rostral ACC is connected to the amygdaloid complex, periaqueductal gray matter, nucleus accumbens, hypothalamus, anterior insula, hippocampus, and the orbitofrontal cortex (Bush et al., 2000; Devinsky et al., 1995), and sends outputs to autonomic, visceromotor, and endocrine systems. It has also been demonstrated recently that microinjections of the excitatory amino acid agonist DL-homocysteic acid into the ACC decreased the innate fearinduced tonic immobility, an effect dependent on glutamatergic neurotransmission in the dorsal periaqueductal gray matter (Coutinho and Menescal-de-Oliveira, 2010). According to some studies, it is possible that thickness abnormalities are a consequence rather than a cause of SP (Rauch et al., 2004). Thus, the thinning of the ACC found in the present work could be a consequence of constitutional and environmental factors. To clarify this uncertainty, longitudinal studies are clearly warranted to provide information concerning functional activation before anxiety symptoms begin to manifest.

Please cite this article as: Linares, I.M.P., et al., Cortical thinning of the right anterior cingulate cortex in spider phobia: A magnetic resonance imaging and spectroscopy study. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.040

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In agreement with our results showing decreased cortical thickness, reduced volumes have been observed in paralimbic cortical regions in OCD (Szeszko et al., 1999), panic disorder (Vythilingam et al., 2000), and post-traumatic stress disorder (Rauch, 2003). These findings support the hypothesis that anxiety disorders share common aspects in their neurobiology, suggesting that our result is not specific to SP. Excessive fear is a component of anxiety disorders. As such, it is an important factor for the comprehension of the neurocircuitry underlying anxiety disorders and fear responses. A set of brain regions have their particular roles in the specific components of fear processing, such as the perception of threat or unconditioned stimuli, and the pairing of an unconditioned stimulus with a conditioned response (Shin and Liberzon, 2001). Regarding the neural correlates of conditioned fear, animal and human studies have identified the amygdaloid complex as a key region within a neuronal network of unconditioned and conditioned fear learning and expression (Buchel et al., 1998; LeDoux, 2000; Phan et al., 2004). The neurocircuitry of fear conditioning and extinction in healthy controls suggests that the amygdaloid complex may be activated during automatic processing of threatening stimuli, and areas such as the insula and cingulate may be subsequently activated (Straube and Miltner, 2006). Recent imaging studies point to the ACC, the insula, the medial prefrontal cortex, the orbitofrontal cortex, and the thalamus as neuronal correlates of conditioned fear responses (Sehlmeyer et al., 2009). Thus, it is possible that the ACC accounts for a share of the threat processing in some anxiety disorders, including SP (Rauch et al., 1995; Osuch et al., 2001; Boshuisen et al., 2002; Pissiota et al., 2002; Carlsson et al., 2004). Most studies that used functional MRI to assess SP populations reported increased ACC activity. Thus, the structural neuroimaging findings reported in the present work are not totally reconcilable with available functional imaging data. This has already been observed in other anxiety disorders such as obsessive compulsive disorder (OCD), with a reduction of the anterior division of the orbitofrontal cortex; whereas previous functional imaging studies demonstrated increased activity in this cortical region (Choi et al., 2004). Choi et al. (2004) also suggested that the increased activity in the orbitofrontal cortex in functional imaging studies may be the result of compensatory hyperactivity of residual tissues secondary to a decreased volume in this cortical area. These observations suggest that increases in regional activity are not necessarily related to increases in volume or thickness, and vice-versa (Ferrari et al., 2008). As a result, structural studies can be complementary to functional approaches, in which the delimitation of anatomical changes, the ability to show that increased or reduced tissue volumes are compatible with hypermetabolism due to compensatory mechanisms, and even suggestion of encephalic structural and functional changes during the course of a given psychiatric disorder (Pujol et al., 2004). Our study has some limitations that deserve mention, including the sample size that may have limited the detection of small effects. The prevalence of female subjects is also a drawback and underscores the need for caution in the generalization of our findings. Nonetheless, most published

5

reports on neuroimaging in animal SP describe findings in female samples. Finally, a warning note not related to this specific study but to all SP neuroimaging studies regards the difficulty in directly translating experimental findings to clinical application. On the other hand, the strengths of our study include its population-based design, the fact that it was the first 1H MRS study to compare metabolite levels in participants with spider SP and healthy controls, the use of a 3 T scan and cortical thickness results, and, finally, its role in helping to elucidate the pathophysiology of SP.

4.

Conclusion

The present results do not support the biochemical involvement of the cingulate cortex in the pathophysiology of SP; however, we documented that there is a thinning of the ACC in spider SP volunteers. Taken together, our findings show the implication of the ACC in the pathophysiology of anxietyrelated neural mechanisms, specifically in SP. These findings may have implications for the understanding of the neuroanatomical circuitry involved in conditioning and innate fear mechanisms, although this hypothesis remains to be confirmed by further studies involving larger samples.

5.

Statistical analysis

The statistical significance level was set at p r0.05. Statistical difference maps between spider SP participants and healthy controls were constructed in FreeSurfer using a GLM vertexby-vertex analysis; corrected for multiple comparisons using false discovery rate (FDR). MRS data and quantitative data were compared using unpaired Student's t-test. Correlations between cortical thickness and symptom scores (BAI, SPQ) and between MRS and symptom scores were calculated using Pearson's test.

6.

Experimental procedures

6.1.

Subjects

Patients with spider phobia (n ¼19) were matched to 17 healthy controls (HC) according to the characteristics summarized in Table 1. Study subjects were recruited via public advertisements in newspapers and TV programs. The spider SP group fulfilled the diagnostic criteria for spider phobia according to the Structured Clinical Interview for DSM-IV (SCID-IV) (Del-Ben et al., 2001; First et al., 1997). None of the participants had a history of neurological, psychiatric or other relevant organic diseases, including substance abuse. Also, none of the participants in the two groups reported the use of prescribed psychotropic medications. The HC and spider SP groups were screened with the Spider Questionnaire (SPQ) (Watts and Sharrock, 1984; Granado et al., 2005) to assess the overall severity – or absence – of spider phobia, and with the Beck Anxiety

Please cite this article as: Linares, I.M.P., et al., Cortical thinning of the right anterior cingulate cortex in spider phobia: A magnetic resonance imaging and spectroscopy study. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.040

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Inventory (BAI) (Beck et al., 1988) to evaluate the severity of general anxiety symptoms. Participants in the spider SP group had significantly higher total scores in the SPQ subscales, as shown in Table 1. Both groups were matched with respect to age, education, and socio-economic status as assessed using Brazilian standardized criteria (Critério de classificação sócio-econômica, 2005). Participants' handedness was determined through the Edinburgh Handedness Inventory (Oldfield, 1971) and only one patient was left-handed. The experimental procedures were approved by the local ethics committee (Ribeirão Preto Medical School, Clinical Research Unit, process number 14554/2009) and written informed consent was obtained from all subjects after a complete description of the study was given. Two of the spider SP subjects presented brain abnormalities in the MRI scan and were therefore excluded from the analysis.

6.2.

Image acquisition

All imaging and spectroscopy data were collected on a 3 T MRI scanner (Siemens, Erlangen, Germany) equipped with 25 mT gradient coils at the Ribeirão Preto Medical School University Hospital. Head movements were restricted with the use of expandable foam cushions. After an automated scout image was acquired, shimming procedures were performed to optimize field homogeneity (Reese et al., 1995). A high-resolution three-dimensional magnetization prepared rapid gradient echo (MPRAGE) was acquired for each subject using the following protocol: TR¼ 9.7, TE¼5 ms, FOV¼ 25.6 cm, matrix 256  256, slice thickness¼ 1 mm, flip angle¼121, yielding 160 slices. After obtaining scout images in the three anatomical planes, the MRS sequence was acquired (TR¼ 1500 ms, TE¼32 ms) based on a single voxel and with a total field of view of 256  256  20 mm3 (length–width–thickness). The volume that was selected for point-resolved spectroscopy (PRESS) excitation to shimming was of 100  80  20 mm3 (length by width by height). The voxels had a total size of 2 mm3 (10  10  20 mm3). The slice was oriented in the cortical region.

6.2.1.

Cortical thickness

Cortical reconstruction, cortical parcellation, and subcortical segmentation were performed using the Freesurfer image analysis package (http://surfer.nmr.mgh.harvard.edu/). This processing method includes removal of non-brain tissue using a hybrid watershed/surface deformation procedure, automated Talairach transformation, segmentation of subcortical white matter and deep gray matter volumes (including the hippocampus), intensity normalization, tessellation of the gray matter/white matter boundary, automated topology correction, and surface deformation following intensity gradients to optimally place the gray/white and gray/cerebrospinal fluid borders at the location where the greatest shift in intensity defines the transition to the other tissue class (for additional details, please see Fischl and Dale, (2000)).

6.2.2.

1

H MRS

The volume of interest (VOI) was the cingulate cortex (see Fig. 1). This region encompasses specialized subdivisions that perform different functions, such as cognitive, emotional, and nociceptive processing (Vogt et al., 1992). Additionally, it seems to play an important role in the neurobiology of SP. 1 H MRS was used to assess the concentrations of N-acetylaspartate (NAA), choline (Cho), and creatine (Cr) in the VOI. Quantification was performed by intravoxel normalization and absolute quantification based on internal measurement of water before its subtraction using the LC Model software package. Post-processing included zero filling up to a 32  32 matrix, a Gaussian filter, and an inverse Fourier transform. The baseline stability criteria ensured that there were no gross asymmetries after adequate eddy current correction. The area under the peak was used as the resonance intensity measure for NAA, Cr, and Cho levels, and these values were calculated using Siemens' spectroscopy software. The output was given as .txt files and transferred to a spreadsheet in Microsoft Excel for the calculation of ratios. Concentrations were then analyzed as ratios between peaks (NAA/Cr and Cho/Cr), and normal values were considered as being within two standard deviations from the means in the HC group.

Conflict of interest statement The authors have no conflict of interest related to the topic of this article. The funding agency had no role in the study design or in the decision to submit the paper for publication.

Acknowledgments We thank the spider phobic and healthy control subjects who Q5 Q6 participated in this study and the research staff. This study was supported by grants from the Fundação de Amparo à Pesquisa de São Paulo (FAPESP). Linares, I.M.P. is the recipient of a FAPESP fellowship (2010–2012). Crippa, J.A.S., Hallak, J.E. C., Zuardi, A.W. and Coimbra, N.C. are recipients of grants from Brazil's Conselho Nacional de Pesquisa (CNPq). Nardi, A. E. is supported by CNPq and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Trzesniak, C. M.F. and Arrais, K.C. are supported by FAPESP. Jackwoski, A.P. is supported by CNPq and FAPESP.

r e f e r e n c e s

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Please cite this article as: Linares, I.M.P., et al., Cortical thinning of the right anterior cingulate cortex in spider phobia: A magnetic resonance imaging and spectroscopy study. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.040

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Please cite this article as: Linares, I.M.P., et al., Cortical thinning of the right anterior cingulate cortex in spider phobia: A magnetic resonance imaging and spectroscopy study. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.040