Focal Gray Matter Density Changes in Schizophrenia FREE

THE VIEW THAT schizophrenia is a brain disease particularly involving decrements in gray matter is supported by findings from many imaging studies.^{1 - 9} Global gray matter volume decreases of approximately 2% were reported in a recent meta-analysis of volumetric magnetic resonance imaging (MRI) studies in schizophrenia.³ However, results from this meta-analysis also suggest that changes may be more prominent in some brain areas than in others.³ Indeed, relative to the cerebral volume differences, volume decreases were found in the anterior superior temporal gyrus (7%), amygdala and hippocampal complex (6%), thalamus (4%), and frontal lobes (2%). Increases relative to the cerebral volume differences were found in the globus pallidus (24%), putamen (6%), and caudate nucleus (4%). Global temporal lobe volume differences, on the other hand, were proportionate to the overall cerebral volume decreases in schizophrenia. Findings from studies using statistical analysis of images on a voxel-by-voxel basis predominantly suggest involvement of the thalamus and prefrontal-thalamic-cerebellar circuitry,^{10 - 11} and structural changes in the temporal pole, insula, amygdala, and dorsolateral prefrontal cortex in schizophrenia.¹² However, diffuse gray (and white) matter abnormalities have also been found.¹³

Recent evidence from longitudinal studies suggests that the decreases in brain volume may be progressive over the course of the illness in schizophrenia,^{14 - 15} and this may also be the case for gray matter changes. A 4-fold greater decrease in cortical gray matter volume and the thalamus area was reported in patients with childhood-onset schizophrenia in a 2-year longitudinal study.^{16 - 17} Synaptic degeneration in the left thalamus with increasing duration of disease was suggested in a neuropathological study in schizophrenia.¹⁸ However, it is unknown whether progressive gray matter loss in schizophrenia affects gray matter globally or whether some areas show more decreases than other areas.

This study analyzed focal gray matter density across a 55-year age span in brain MRI of 159 patients with schizophrenia or a schizophreniform disorder and 158 healthy subjects using voxel-based morphometry. Voxel-based morphometry has several advantages over the standard volume measurements. Using this technique, several studies have demonstrated structural brain abnormalities among different patient populations.^{19 - 22} Voxel-based morphometry is not biased to one particular structure, includes structures that are difficult to quantify by the standard volume measurements, and provides a comprehensive assessment of anatomical differences throughout the brain.²³ The aim of this study was to examine the presence of focal changes in gray matter density in schizophrenia and, if present, whether these become more pronounced with age.

One hundred fifty-nine patients with schizophrenia or schizophreniform disorders and 158 healthy comparison subjects from the Utrecht Schizophrenia Project, Utrecht, the Netherlands, participated after written informed consent was obtained. All subjects were between the ages of 16 and 70 years. Subjects with a major medical or neurological illness including migraine, epilepsy, hypertension, cardiac disease, diabetes mellitus, endocrine disorders, cerebrovascular disease, alcohol or other drug dependence, head trauma in the past, or an IQ below 80 were excluded. To allow for unbiased estimates of gray matter density changes with age in patients, care was taken that the severity of illness was evenly distributed across the age range in patients. The number of patients who were recruited from various outpatient and inpatient clinics was evenly distributed across the age range. Patient outcome (defined by the logarithmically transformed ratio of the cumulative months of hospitalization and the cumulative months of illness since first symptoms) was not statistically significantly correlated with their age (r = 0.01, P = .88).

The presence or absence of psychopathological abnormality was established in all subjects using the Comprehensive Assessment of Symptoms and History²⁴ and Schedule for Affective Disorders and Schizophrenia Lifetime Version²⁵ assessed by trained and experienced psychologists and psychiatrists (H.E.H., N.E.M.H., H.K., and colleagues) in the Department of Psychiatry, University Medical Center, Utrecht. Diagnostic consensus was achieved in the presence of a senior psychiatrist (R.S.K. and colleagues). All patients met DSM-IV diagnoses of schizophrenia or schizophreniform disorder; those with schizophreniform disorder met the criteria for the diagnosis of schizophrenia after 1 year of illness. All healthy comparison subjects met Research Diagnostic Criteria²⁶ of "never [being] mentally ill."

Age at onset of illness was defined as the first time patients had been seeking medical or psychological help for their psychotic symptoms. All patients had received antipsychotic medication in the past and all but 4 patients received antipsychotic medications at the time of the MRI scan. Medication included typical and atypical (clozapine, risperidone, olanzapine, or sertindole) antipsychotic agents. Medication dose was expressed as haloperidol-dose equivalents. Subjects were matched for age, sex, and height, and for the socioeconomic status of their parents expressed as the highest completed level of education by one of their parents. There were no interaction effects of age by sex for patients compared with controls or for age at onset by sex within the patient cohort (Table 1).

Brain MRI scans from all subjects were evaluated by 2 independent clinical neuroradiologists. No gross abnormalities were reported in any of the subjects.

Magnetic resonance images were acquired using a scanner (Philips Gyroscan; Philips Medical Systems, Best, the Netherlands) operating at 1.5 T in all subjects. T1-weighted, 3-dimensional, fast field echo scans with 160 to 180 1.2-mm contiguous coronal slices (echo time [TE], 4.6 milliseconds; repetition time, 30 milliseconds; flip angle, 30°; field of view, 256 mm; and in-plane voxel sizes, 1 × 1 mm²) and T2-weighted, dual echo turbo spin echo scans with 120 1.6-mm contiguous coronal slices (TE1, 14 milliseconds; TE2, 80 milliseconds; repetition time, 6350 milliseconds; flip angle, 90°; field of view, 256 mm; and in-plane voxel sizes, 1 × 1 mm²) of the whole head were used for quantitative measurements. In addition, T2-weighted, dual echo turbo spin echo scans with 17 axial 5-mm slices and a 1.2-mm gap (TE1, 9 milliseconds; TE2, 100 milliseconds; flip angle, 90°; field of view, 250 mm; and in-plane voxel sizes, 0.98 × 0.98 mm²) were used for clinical neurodiagnostic evaluation. Processing was done on the neuroimaging computer network of the Department of Psychiatry, which includes workstations (Unix 9000; Hewlett Packard, Palo Alto, Calif), a computer server, and Pentium III–equipped personal computers. Prior to quantitative assessments 10 MRIs were randomly chosen and cloned for intrarater reliability determined by the intraclass correlation coefficient. All MRIs were coded to ensure masking for subject identification and diagnosis, scans were put into a Talairach frame (no scaling), and corrected for inhomogeneities in the magnetic field.²⁷ Binary masks of gray matter were made based on histogram analyses and a series of mathematical morphological operators to connect all voxels of interest within the cranium, as validated previously.²⁸ The binary gray matter masks were then analyzed using voxel-based morphometry. The binary gray matter masks were resampled to a voxel size of 2 × 2 × 2.4 mm³, blurred using an isotropic Gaussian kernel (full width at half maximum of 8 mm) to generate gray matter "density maps." The density maps represent the local concentration of gray matter (between 0 and 1) per voxel. Each of the MRIs was transformed into a standardized coordinate system in a 2-stage process using the ANIMAL algorithm.²⁹ In the first step, a linear transformation was found by minimizing a mutual information joint entropy objective function computed on the gray level images.³⁰ A nonlinear transformation was computed in the second step by maximizing the correlation of the subject's image with that of a standardized brain. The nonlinear transformation is run up to a scale (full width at half maximum of 4 mm) that aligns global anatomical regions while minimally affecting local volume changes. The standardized brain was selected earlier among 200 brain MRIs of healthy subjects between the ages of 16 and 70 years. To select the standardized brain, all 200 brain MRIs were registered to the Montreal standard brain³¹ and averaged, yielding one average brain image. The mean square error on the normalized intensity values was computed between each of the brain MRIs and the average brain image. The standardized brain was the brain image with the smallest mean square error. Transformations were then applied to the gray matter density maps to remove global differences in the size and shape of individual brains.

Linear regression analysis was performed through all brains for each voxel separately in the gray matter density maps (other examples of this procedure^{32 - 34} ). Group (schizophrenia and healthy comparison subjects), sex (male and female), and handedness (right, left, or ambidextrous) entered the analysis as predictor variables; age served as a covariant. To evaluate the changes with age in schizophrenia, a similar linear regression analysis was performed, adding the interaction between age and schizophrenia as predictor variable. Given the number of subjects, data resolution, voxel size, and volume of the search region, the critical threshold t value for a 2-tailed α significance level of P<.05 after correcting for multiple comparisons is |t|>5.0, according to random field theory.³⁵ For the main effects of schizophrenia, effects at |t|>5.0 were provided in Table 2 and Figure 1. Note that for interaction effects with age, effects at |t|>4.5 were provided in Table 3, Figure 2, and Figure 3 to show that significant (at |t|>5.0) age-related changes represented a cluster of voxels.

In case of a significant main or interaction effect between age and schizophrenia, post hoc analyses were done on local minima and maxima—these were voxels that represented the local minimum or maximum (of a cluster of significant voxels) in the t-map: (1) adding antipsychotic medication dose or outcome as predictor variable to the regression analysis in the patients; (2) (for interaction effects with age only) adding the SD of the unstandardized residual of the dependent variable per age decade as weight in weighted linear regression analysis to exclude cohort biasing effects of the cross-sectional design; and (3) adding time of the scan to the analysis and calculating partial Pearson product moment correlation coefficients correcting for age to exclude time of measurement artifacts.

Linear regression analysis revealed significant decreases in gray matter density in patients with schizophrenia compared with healthy comparison subjects in the left amygdala; left hippocampus; thalamus; (orbito) frontal, temporal, occipitotemporal, precuneate, posterior cingulate, and insular gyri bilaterally (Figure 1A-C and Table 2). In patients with schizophrenia significant increases in gray matter density were found exclusively in the right caudate nucleus and globus pallidus (Figure 1D and Table 2).

A significant interaction with age was found for gray matter density in the left amygdala in patients with schizophrenia (t = −5.01, unstandardized regression coefficient b = −0.078 density change over 55 years, reflecting approximately 8% density loss); the density of the left amygdala did not decrease significantly with age in healthy comparison subjects (Figure 2, Figure 3, and Table 3).

Age at onset, outcome, antipsychotic medication at the time of the MRI scan acquisition, and interactions with sex and age or age at onset did not explain these gray matter density changes in patients with schizophrenia. Moreover, the SD of the unstandardized residual of gray matter density per age decade and time of the MRI scan did not explain the findings.

This cross-sectional study compared gray matter density in 159 patients with schizophrenia or a schizophreniform disorder and 158 healthy comparison subjects across a 55-year age span. Its main finding is that distinct focal areas in the brains of patients with schizophrenia or a schizophreniform disorder display decreased gray matter density, including the left amygdala; left hippocampus; right supramarginal gyrus; thalamus; (superior) temporal, occipitotemporal, precuneate, posterior cingulate, and insular gyri bilaterally. Moreover, the left amygdala density decrease was more pronounced in the older than in the younger patients with schizophrenia. These findings could not be explained by outcome, age at onset, antipsychotic medication at the time of the MRI scan, interactions of sex with age or age at onset, changes in gray matter density variation with age, or by time of measurement effects.

Significant changes in gray matter density were found in brain regions that were previously reported to be altered in volume in schizophrenia.^{1 - 9 ,36 - 42} The primarily left-sided involvement of the medial temporal area is in agreement with suggested decreased hemispheric dominance with predominantly left-sided brain involvement in schizophrenia,⁴³ although the thalamic and other (cortical) decreases were bilateral. Thalamic decreases have been reported in several studies.^{10 ,36} Close inspection of the local minimum of the t-map of the left and right thalamus in Table 2 and of Figure 1B further suggests that the decreases may be particularly pronounced in the dorsomedial nucleus, which is consistent with recent evidence of a volume reduction in this nucleus in (first-episode) schizophrenia^{44 - 45} and with a loss of neurons in this nucleus in the brains of schizophrenic patients in postmortem studies.^{46 - 47} Interestingly, the mediodorsal nucleus of the thalamus has prominent anatomical connections with the frontal cortex, including the orbitofrontal cortex,⁴⁸ where highly significant gray matter density decreases were found in our study. These density decreases are in agreement with the decreased orbitofrontal gray matter volume reported earlier in schizophrenia.^{6 ,38} Decreased orbitofrontal gray matter has been related to increased negative symptoms in male⁴⁹ and female³⁸ patients with schizophrenia. Also, in a postmortem study decreases of D₃ and D₄ dopamine receptor transcripts, suggestive for a disruption of cortical dopaminergic neurotransmission at the level of receptor expression, were found to be restricted to the orbitofrontal cortex of patients with schizophrenia.⁵⁰ The gray matter density decreases in the middle and medial superior frontal areas are in concordance with decreased volumes in these areas of the frontal cortex found previously (although only in male patients).³⁷ They furthermore support functional MRI studies suggesting aberrant frontal cortex activation during working memory tasks in schizophrenia.^{51 - 52} Temporal lobe involvement in our study was predominantly found in the (anterior) superior temporal gyri bilaterally, which is consistent with findings from volumetric studies.³ Increases in gray matter density were exclusively found in the (right) caudate and globus pallidus nuclei that are part of the basal ganglia. These nuclei were found to be increased in volume in several earlier studies in patients with schizophrenia who were taking (typical) antipsychotic medication.^{39 - 41}

Additional areas with significant decreases in gray matter density in schizophrenia were the insula, posterior cingulate, precuneate, supramarginal gyrus, and occipitotemporal gyri. These structures have not been extensively reported on in volumetric studies in schizophrenia. Involvement of the insula in schizophrenia was suggested earlier in a few volumetric studies^{6 ,53} and in a study using voxel-based morphometry.¹² Anatomically, connections between the insula and the amygdala; thalamus; and (orbito) frontal, (superior) temporal, and cingulate gyri have been described.⁵⁴ Functionally, the insula is implicated in somatosensory processing, showing increased activation during pleasant and aversive taste stimuli.⁵⁵ It is part of a pain perception network⁵⁶ that may also include a (separate) area that activates during the anticipation of pain.⁵⁷ Interestingly, sensitivity to pain appears diminished in patients with schizophrenia⁵⁸ and in a few of their relatives.⁵⁹ In addition, the insula may be involved in conveying a cortical representation of fear to the amygdala.⁶⁰ The posterior cingulate also deserves further study in schizophrenia. It is part of the retrosplenial cortex which, on the basis of functional imaging studies, is considered to play a prominent role in the processing of emotionally salient stimuli.⁶¹ The gray matter density decreases in the precuneus, or medial parietal cortex, may be associated with the aberrant activation pattern in this area in the brains of patients with schizophrenia during verbal fluency⁶² and long-term memory recognition.⁶³ Involvement of the supramarginal gyrus has been reported before and may be related to attentional abnormalities in patients. The small areas of decreased gray matter density in the medial occipitotemporal gyrus, or lingual gyrus, were reported earlier in a voxel-based morphometric study.¹¹ This area has been associated with spatial information processing,⁶⁴ which may be impaired in schizophrenia.⁶⁵ In a volumetric MRI study the posterior cingulate, precuneate, and occipitotemporal gyri were not significantly decreased in schizophrenia.⁶ This may not be in contrast to our findings, however, since the areas of decreased gray matter density in these brain regions were rather small and may, therefore, not have reached significance in the larger parcellation units of the cortex as defined in the volumetric study.

The more prominent decrease in left amygdala density in older compared with younger patients with schizophrenia suggests a loss of gray matter in this limbic brain region over the course of the illness. This finding is consistent with the theory that neurodegenerative brain changes such as progressive dysplasia at the synaptic or cellular level may be involved in schizophrenia,⁶⁶ possibly in addition to neurodevelopmental processes. In healthy subjects no changes with age were found in the amygdala, which is consistent with earlier findings suggesting that the volume of the amygdala starts to decrease only after the age of 60 years.⁶⁷ However, a cohort effect might also explain the interaction between age and diagnosis. Since the amygdala has particularly widespread anatomical projections to the lateral orbitofrontal gyri⁶⁸ and hippocampus—as well as to the medial orbitofrontal gyri, insula, temporal pole, and mediodorsal nucleus of the thalamus,⁶⁹ while receiving extensive sensory input from the neocortex⁷⁰ —our findings may suggest that a circuit involving the amygdala and its projection areas show (progressive) changes in schizophrenia. Interestingly, meta-analysis of hippocampal complex studies in schizophrenia found that inclusion of the amygdala in the region of interest significantly increased effect sizes across studies,⁴² providing indirect evidence for decreased amygdala volume in schizophrenia. Neuropathological studies revealed extensive loss of tissue⁷¹ and an increase in dopamine⁷² and its metabolite homovanillic acid⁷³ in the left amygdala in schizophrenia. Moreover, patients with schizophrenia showed aberrant activation of the left amygdala during exposure to pictures of happy and sad facial expressions.⁷⁴ Indeed, involvement of the amygdala in schizophrenia would be consistent with its central role in emotional and social behavior⁷⁵ —both of which are disturbed in schizophrenia. It would also be consistent with a functional disconnection syndrome in schizophrenia, induced by aberrant modulation of synaptic plasticity in the context of emotional and social learning, as has been suggested previously.⁷⁶

This study was based on in vivo MRIs of 159 brains of patients with schizophrenia or a schizophreniform disorder and 158 healthy comparison subjects using voxel-based morphometry. These measurements do not allow for inferences into the mechanisms that may be involved in this process, which remain to be elucidated in future (postmortem) studies. Moreover, the patients' medication may have influenced findings.⁷⁷ Although antipsychotic medication taken at the time of the MRI scan was unrelated to the age-associated decrease in left amygdala density, effects of cumulative medication cannot be ruled out. Effects from cohort or time of measurement cannot be excluded completely.⁷⁸ Yet, it seems unlikely that they explained the findings, as possible confounding effects of outcome, age at onset of the disease, changes in variance with age, and time of the scan did not influence the results. In future studies, more refined analyses may relate the focal gray matter density changes to the heterogeneity of the disease symptoms in these patients,⁷⁹ as was suggested before.⁸⁰

This study found distinct focal areas in the brains of patients with schizophrenia with decreased gray matter density, including the left amygdala; left hippocampus; right supramarginal gyrus; thalamus; and the (orbito) frontal, (superior) temporal, and occipitotemporal precuneate, posterior cingulate, and insular gyri bilaterally. Moreover, the decreased density in the left amygdala was more prominent in older patients with schizophrenia or a schizophreniform disorder.

Accepted for publication June 26, 2001.

This research was supported by grant 7F99.(2).37 from the HersenStichting Nederland (Dutch Brain Foundation), The Hague, the Netherlands (Dr Hulshoff Pol).

Correponding author and reprints: Hilleke E. Hulshoff Pol, PhD, Department of Psychiatry, A01.126 University Medical Center, Utrecht, 3584 CX Utrecht, the Netherlands (e-mail: h.e.hulshoff@psych.azu.nl).