In Vivo Reactive Astrocyte Imaging in Patients With Schizophrenia Using Fluorine 18–Labeled THK5351

Key Points Question Can region-specific reactive astrocytes in vivo associated with positive symptoms in patients with schizophrenia be measured using fluorine 18–labeled THK5351 ([18F]THK5351) positron emission tomography? Findings In this case-control study of 68 participants, standardized uptake value ratios (SUVrs) of [18F]THK5351 in the bilateral anterior cingulate cortex and left hippocampus were greater in patients with schizophrenia than in healthy controls. Increased [18F]THK5351 SUVrs were correlated with positive symptom severity in patients with schizophrenia. Meaning Considering the role of astrocytes in brain development, neurotransmission, and immune reactions, reactive astrocytes in the bilateral anterior cingulate cortex and left hippocampus may be strong biomarkers for schizophrenia pathophysiology and treatment.

the bilateral anterior cingulate cortex and left hippocampus were greater in patients with schizophrenia than in healthy controls.Increased

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Introduction
Glutamatergic imbalance and neuroinflammation are believed to be important in schizophrenia pathophysiology.2][3] Furthermore, excessive glutamate activity might lead to excitotoxic damage and oxidative stress-related neuroinflammation, which further explains the pathophysiology. 4,51][12][13] Among individuals at clinically high risk for psychosis, the baseline hippocampal glutamate level was suggested to be associated with the transition to psychotic disorder. 146][17][18] These inconsistencies may be due to the simple approach of using individual glutamate or neuroinflammation markers in a rather complex, mutually influencing system.However, investigations aimed at integrating glutamate imbalance and neuroinflammation in the pathophysiology of schizophrenia are limited, and only 1 study 5 has attempted to bridge the gap between the 2 hypotheses by showing that the levels of the antioxidant glutathione and excitotoxic glutamate and/or glutamine are lower in patients with schizophrenia who are in stable clinical condition.
0][21] Reactive astrocytes are remodeled in response to injury, disease, or infection of the brain and can be measured in vivo by detecting overexpressed monoamine oxidase B (MAO-B) on the outer mitochondrial membrane. 22In patients with schizophrenia, abnormal astrocyte-neuronal interactions have been suggested to be the mechanism of psychotic symptom development, 2 and alterations in the expression of astrocyte-related genes and their products in patients' postmortem brains have been reported. 23However, in vivo reactive astrocyte imaging has not yet been reported in patients with schizophrenia.
In this study, we investigated the in vivo imaging of reactive astrocytes and their association with positive symptoms in patients with schizophrenia using validated MAO-B-binding fluorine 18 ([ 18 F])-labeled THK5351 PET [24][25][26] to obtain a more comprehensive understanding of the role of reactive astrocytes in schizophrenia pathophysiology.The primary regions of interest (ROIs) were the ACC and hippocampus based on previous studies of glutamate imbalance, neuroinflammation, and positive symptom development in patients with schizophrenia. 10,13,16,27The secondary ROIs included other limbic regions, such as the posterior cingulate cortex (PCC), parahippocampal gyrus, amygdala, insula, and nucleus accumbens, based on previous studies 28,29 that reported the association between glutamate alterations and positive symptoms in these regions in patients with schizophrenia.

Methods
This case-control study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.All participants provided written informed consent

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In Vivo Reactive Astrocyte Imaging in Patients With Schizophrenia Using [ 18 F]THK5351 after receiving a thorough explanation of the study procedure.The study was conducted in accordance with the Declaration of Helsinki 30 and was approved by the Institutional Review Board of Seoul National University Hospital.-Y.M., and S.K.L.).Psychotic symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS). 31The Hamilton Rating Scale for Depression 32 and the Hamilton Rating Scale for Anxiety 33 were used to measure the severity of depression and anxiety, respectively.The healthy controls were recruited via internet advertisement and were screened using the SCID-I Nonpatient Edition.Healthy controls were excluded if they had any past or current diagnosis of a psychiatric disorder and any first-to third-degree biological relatives with a psychotic disorder.In all participants, general functional status was evaluated using the modified Global Assessment of Functioning, and intelligent quotient (IQ) was measured with the Korean version of the Wechsler Adult Intelligence Scale. 34The exclusion criteria included substance abuse or dependence (except nicotine), neurological disease or significant head trauma, medical illness that could accompany psychiatric symptoms, and intellectual disability (IQ < 70).

PET-MR Image Acquisition
A PET-MR machine (Biograph mMR; Siemens Healthcare) was used to obtain dynamic 3-dimensional PET images.Immediately after an intravenous bolus injection of 185 MBq (5 mCi) of [ 18 F]THK5351, 27 frames of emission scans (8 × 15 seconds, 3 × 60 seconds, 5 × 120 seconds, and 11 × 300 seconds) and a total 70-minute PET scan were acquired while the participant was at rest.Fluorine 18-labeled THK5351 was synthesized and radiolabeled at Seoul National University Hospital, and details are provided in the eMethods in Supplement 1.Each participant was fitted with an MR imaging coil and supporting cushion to reduce head motion during the PET scan, and the participants were asked to remain as still as possible during the scan.
Manufacturer's software from the PET-MR device (e7tool; Siemens Healthcare) was used for the reconstruction of the PET data.The PET images were reconstructed using the ordered-subset expectation maximization algorithm with 24 subsets and 5 iterations.Images were filtered with a 4-mm full-width at half-maximum Gaussian filter at the center of the field of view (image matrix, 256 × 256; 127 sections; voxel size, 1.4 × 1.4 × 2.0 mm).Segmentation-based attenuation correction was conducted with a 3-tissue segmentation map acquired by an ultrashort echo time (TE) sequence (repetition time [TR], 11.9 milliseconds; TE 1, 0.07 milliseconds; TE 2, 2.46 milliseconds; flip angle, 10°; 192 × 192 matrix).A high-resolution structural T1 image (TE, 2.2 milliseconds; TR, 2400 milliseconds; flip angle, 8°; 0.85-mm section thickness) was also collected for each participant at the same time to rule out structural lesions in the brain and to provide an anatomical reference for the [ 18 F]THK5351 analysis.

PET Image Analysis
All preprocessing was conducted using Statistical Parametric Mapping (SPM 12; Welcome Department of Imaging Neuroscience).The analysis flowchart and selected cerebellar lobules are presented in eFigure 1 in Supplement 1.
For semiquantitative PET analysis, standardized uptake value ratios (SUVrs) were calculated in reference to the inferior cerebellar parcels of the cerebellar cortex.To delineate the inferior cerebellar

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In Vivo Reactive Astrocyte Imaging in Patients With Schizophrenia Using [ 18 F]THK5351 ROI, the SUIT (spatially unbiased infratentorial template) toolbox, 35 which contains a high-resolution atlas template of the cerebellum and brainstem and individual T1 images, was used.The cerebellar structure was isolated from the cerebral structure and segmented into tissue types using the Dartel algorithm.Using the deformation field obtained during the Dartel procedure, the SUIT template was transferred to an individual PET space.The lobular ROIs that corresponded to the inferior cerebellar gray matter (bilateral Crus II, VIIb, VIIIa, VIIIb, and IX) were used to extract radioactivity from the PET image.After frame-by-frame motion correction of the PET image, the T1 images were coregistered to the mean images of 27 realigned PET frames.
The bilateral ACC and hippocampus, as primary ROIs, and other limbic regions (bilateral PCC, parahippocampal gyri, amygdala, insula, and nucleus accumbens), as secondary ROIs, were predefined using the Wake Forest University PickAtlas toolbox in SPM 12. 36 The predefined ROIs were transformed into PET standard space using the deformation matrix calculated from PET-coregistered T1 images, and SUVs were extracted for all PET frames to assess the time activity curve in each ROI.Finally, based on previous studies that tested the optimal time windows to estimate [ 18 F]THK5351 quantification, 37 the SUVr was calculated as the sum of 45-to 65-minute postinjection frames using the mean radioactivity of the inferior cerebellar ROI obtained by the SUIT procedure as a reference.

Statistical Analysis
Data were collected from October 1, 2021, to January 31, 2023.Demographic and clinical characteristics were compared between patients with schizophrenia and healthy controls using an independent t test or a Welch t test if the variance was not equal and a χ 2 test or a Fisher exact test for categorical data.Group differences in the SUVr in the bilateral ACC and hippocampus (ie, primary ROIs) were tested using analysis of covariance, with age and sex as covariates.Pearson correlation analysis was performed to investigate the association between altered SUVrs in primary ROIs and PANSS positive symptom scores in patients with schizophrenia.To rule out the possible effect of the duration of illness or olanzapine-equivalent dose of antipsychotics prescribed at the time of study participation on the SUVrs of the primary ROIs, Pearson correlation analysis was performed.To account for multiple comparisons, a false discovery rate (FDR) correction was performed.Group differences in SUVrs in other limbic ROIs were assessed using repeated-measures analysis of variance (ANOVA), with brain regions (ie, bilateral PCC, parahippocampal gyri, amygdala, insula, and nucleus accumbens) as the within-participants factor and age and sex as covariates.All statistical analyses were performed in SPSS, version 25.0 for Windows (IBM Corporation), and the level of statistical significance was set at 2-sided P < .05.  1 summarizes the demographic and clinical characteristics of the participants.There were no significant group differences in age or sex, while mean (SD) IQ (107.2Supplement 1).Repeated-measures ANOVA revealed trend-level group differences in the SUVrs in the secondary ROIs (eg, right parahippocampal gyrus, F = 3.387 [P = .07])(eFigure 2 and eTable 2 in Supplement 1).There was no correlation between the duration of illness or olanzapine-equivalent dose of antipsychotics prescribed at the time of study participation and the SUVrs of the primary ROIs (eTable 3 in Supplement 1).

Discussion
In this study, we investigated in vivo reactive astrocyte imaging using MAO-B-binding [ 18 F]THK5351 PET to reveal the role of reactive astrocytes, which are involved in both glutamate imbalance and neuroinflammation, in schizophrenia pathophysiology.Patients with schizophrenia had elevated SUVrs in the bilateral ACC and left hippocampus compared with healthy controls.In addition, positive correlations between the SUVrs in the bilateral ACC and the PANSS-positive symptom scores were found in patients with schizophrenia.There were trend-level group differences in the SUVrs in other limbic regions investigated as secondary ROIs.The results of this study not only provide in vivo neuroimaging evidence of the role of reactive astrocytes in schizophrenia pathophysiology but also highlight the region-specific association between reactive astrocytes in the ACC and positive symptoms in patients with schizophrenia.
Our findings of elevated MAO-B-binding [ 18 F]THK5351 uptake in patients with schizophrenia suggest a role for reactive astrocytes in the neurodevelopmental abnormalities of these patients.
Astrocytes are known to be important in neurodevelopment and to play a critical role in synapse c Statistically significant at P < .005.
d Scores range from 30 to 210, with higher scores indicating greater severity of psychotic symptoms.
e Scores range from 0 to 56, with higher scores indicating greater severity of anxiety.
f Scores range from 0 to 52, with higher scores indicating greater severity of depression.
g Scores range from 1 to 100, with higher scores indicating higher functioning.
h Mean olanzapine equivalent dose of antipsychotics prescribed at the time of study participation.b Analysis of covariance with age and sex as covariates.
c Statistically significant at P < .05.

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In formation and function as well as neuronal survival and migration; thus, abnormal astrocytes can increase the vulnerability of the brain to neurodevelopmental disorders such as schizophrenia. 38,39ndrem et al 40 showed that mice chimerized with induced pluripotent stem cells derived from patients with childhood-onset schizophrenia exhibited problems with glial cells, including astrocytes, suggesting that genetic abnormalities in schizophrenia produce abnormalities in astrocytes, which are critical for brain development and schizophrenia pathophysiology.Thus, the results of the present study support the vulnerability and neurodevelopmental model of schizophrenia and suggest the role of reactive astrocytes in schizophrenia pathophysiology by providing in vivo neuroimaging evidence of reactive astrocytes in patients with schizophrenia.
Furthermore, the results of this study provide integrative supporting evidence for the glutamate and immune hypotheses in schizophrenia pathophysiology.Previous studies aimed at measuring glutamate and/or glutamine levels in patients with schizophrenia 11,13 have produced inconsistent results, which may be due to the lack of consideration of interacting systems other than glutamate.
Microglial imaging studies using translocator protein PET to show neuroinflammation in patients with schizophrenia 16,18 have also provided insufficient evidence that may be caused by subtle changes in microglial activity.Astrocytes not only play important roles in glutamate cycling and synaptic transmission but also undergo astrogliosis in reaction to neuroinflammation, as shown in neuroinflammatory diseases such as Wilson disease and multiple sclerosis. 2,6,25,41Therefore, the results of the present study support that both the glutamate and immune hypotheses can provide a more comprehensive understanding of the interaction between glutamate and the immune system in the pathophysiology of schizophrenia by revealing reactive astrocytes in vivo in patients with schizophrenia.
In the present study, elevated [ 18 F]THK5351 uptake was detected in the bilateral ACC and left hippocampus of patients with schizophrenia compared with healthy controls.These findings are consistent with previous studies 23,42 reporting that elevated astrocyte-, glutamate-, and immunerelated genes and gene products were found in the postmortem brains of patients with schizophrenia.Previous neuroimaging studies targeting the glutamate system or neuroinflammation 13,14,16,28,43 have also reported alterations in the ACC and hippocampus, which are known to be important brain regions involved in schizophrenia.In addition, an elevated SUVr of MAO-B-binding [ 18 F]THK5351 in the bilateral ACC was positively correlated with positive symptom severity, as measured by the PANSS, in patients with schizophrenia.The ACC is one of the limbic cortices, and its role in fundamental cognitive processes, such as motivation, decision-making, and social cognition, which are impaired in patients schizophrenia in relation to psychotic symptoms, has been highlighted. 44,45A previous study from Kim et al, 46 which revealed that thalamocortical dysrhythmia represented by elevated resting-state theta phase-gamma amplitude coupling in the ACC of patients with schizophrenia spectrum disorder was positively correlated with symptom severity, also supported the association of reactive astrocytes in the ACC with positive symptoms. 2

Limitations
This study has several limitations.First, the current study participants were patients with schizophrenia who had more than 5 years of antipsychotic treatment, although there have been suggestions of an association between prolonged exposure to antipsychotics and increased MAO-B expression in animal studies. 47,48However, there have been no reports regarding the association between chronic antipsychotic exposure and MAO-B elevation in patients with schizophrenia, and a recent study 49 suggested that MAO-B detected by PET imaging is a target for novel drug development in patients with schizophrenia. 23,50In addition, we did not find any correlation between the duration of illness or olanzapine-equivalent dose of antipsychotics prescribed at the time of study participation and the SUVrs of the primary ROIs.The current study results should be interpreted with caution because we did not perform this study in patients who were drug naive or with first-episode psychosis who may experience a minimal effect of antipsychotics on MAO-B Pearson correlation analysis between standardized uptake value (SUV) ratios of the bilateral anterior cingulate cortex (ACC) and positive symptom scores on the Positive and Negative Syndrome Scale (PANSS; range, 7-49, with higher scores indicating the greater severity of positive symptoms) was performed.Statistical significance was determined as a false discovery rate (FDR)-corrected P < .05.
The orange lines indicate regression lines.

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In Vivo Reactive Astrocyte Imaging in Patients With Schizophrenia Using [ 18 F]THK5351 expression.Second, because of the limitations of the MAO-B-detecting PET method, this study could only provide indirect evidence of reactive astrocytes in schizophrenia pathophysiology, as in previous animal, postmortem brain, and neuroimaging studies. 13,21,23,40Further studies with direct methods to investigate astrocytes in patients with schizophrenia are warranted to confirm the role of reactive astrocytes in the pathophysiology of schizophrenia.Third, the results of the present study are exploratory findings that need further validation in other studies due to the small effect size associated with the small sample size and other limitations mentioned above.

Conclusions
This case-control study provides novel in vivo imaging evidence of reactive astrocyte involvement in the pathophysiology of schizophrenia.Considering the role of astrocytes in brain development, neurotransmission, and immune reactions, reactive astrocytes can be a strong biomarker for schizophrenia treatment.In particular, reactive astrocytes in the ACC may be a future target of neuromodulation therapeutics for the positive symptoms of schizophrenia.To support the findings of the present study, a more direct investigation of astrocytes generated by the reverse differentiation of induced pluripotent stem cells derived from patients with schizophrenia is needed.

[ 18 F
]THK5351 SUVrs were correlated with positive symptom severity in patients with schizophrenia.Meaning Considering the role of astrocytes in brain development, neurotransmission, and immune reactions, reactive astrocytes in the bilateral anterior cingulate cortex and left hippocampus may be strong biomarkers for schizophrenia pathophysiology and treatment.

A
total of 33 patients with schizophrenia and 35 age-and sex-matched healthy controls participated in this study.All study participants were of East Asian descent.Information regarding the sample size calculation is provided in the eMethods in Supplement 1. Patients with schizophrenia were recruited from the outpatient office of the Department of Neuropsychiatry at Seoul National University Hospital.The diagnosis of schizophrenia was confirmed using the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) Axis I Disorders (SCID-I) by board-certified psychiatrists (M.K., S.

a
Data are multiplied by 10 −3 .

Figure 2 .
Figure 2. Correlation Between Fluorine 18-Labeled THK5351 Retention and Positive Symptom Severity in Patients With Schizophrenia

Table 1 .
Demographic and Clinical Characteristics of the Participants a Unless otherwise indicated, data are expressed as mean (SD).bCalculated using an independent t test or a Welch t test if the variances were not equal, and a χ 2 test or a Fisher exact test for categorical data.

Table 2 .
Group Comparison Results of Fluorine 18-Labeled THK5351 Uptake in Primary ROIs Abbreviations: FDR, false discovery rate; ROI, region of interest; SUV, standardized uptake value.