Generalization of endothelial modelling of TSPO PET imaging: Considerations on tracer affinities.

The 18 kDa translocator protein (TSPO) is a marker of microglia activation and the main target of positron emission tomography (PET) ligands for neuroinflammation. Previous works showed that accounting for TSPO endothelial binding improves PET quantification for [11C]PBR28, [18F]DPA714 and [11C]-R-PK11195. It is still unclear, however, whether the vascular signal is tracer-dependent. This work aims to explore the relationship between the TSPO vascular and tissue components for PET tracers with varying affinity, also assessing the impact of affinity towards the differentiability amongst kinetics and the ensuing ligand amenability to cluster analysis for the extraction of a reference region. First, we applied the compartmental model accounting for vascular binding to [11C]-R-PK11195 data from six healthy subjects. Then, we compared the [11C]-R-PK11195 vascular binding estimates with previously published values for [18F]DPA714 and [11C]PBR28. Finally, we determined the suitability for reference region extraction by calculating the angle between grey and white matter kinetics. Our results showed that endothelial binding is common to all TSPO tracers and proportional to their affinity. By consequence, grey and white matter kinetics were most similar for the radioligand with the highest affinity (i.e. [11C]PBR28), hence poorly suited for the extraction of a reference region using supervised clustering.

A Unified Framework for Plasma Data Modeling in Dynamic Positron Emission Tomography Studies.

OBJECTIVE: Full quantification of dynamic positron emission tomography (PET) data requires the knowledge of tracer concentration in the arterial plasma. However, its accurate measurement is challenging due to the presence of radiolabeled metabolites and measurement noise. Mathematical models are fitted to the plasma data for both radiometabolite correction and data denoising. However, the models used are generally not physiologically informed and not consistently applied across studies even when quantifying the kinetics of the same radiotracer, introducing methodological variability affecting the results interpretation. The aim of this study was to develop and validate a unified framework for the arterial data modeling to achieve an accurate and fully automated description of the plasma tracer kinetics. METHODS: The proposed pipeline employs basis pursuit techniques for estimating both radiometabolites and parent concentration models from the raw plasma measurements, allowing the resulting algorithm to be both robust and flexible to the different quality of data available. The pipeline was tested on four PET tracers ([11C]PBR28, [11C]MePPEP, [11C]WAY-100635, and [11C]PIB) with continuous and discrete blood sampling. RESULTS: Compared to the standard procedure, the pipeline provided similar fit of the parent fraction but yielded a better description of the total plasma radioactivity, which in turn allowed a more accurate fit of the tissue PET data. CONCLUSION: The new method showed superior fits compared to the standard pipeline, for both continuous and discrete arterial sampling protocol, yielding to better description of PET data. SIGNIFICANCE: The proposed pipeline has the potential to standardize the blood data modeling in dynamic PET studies given its robustness, flexibility and easiness of use.

The effects of haloperidol on microglial morphology and translocator protein levels: An in vivo study in rats using an automated cell evaluation pipeline.

BACKGROUND: Altered microglial markers and morphology have been demonstrated in patients with schizophrenia in post-mortem and in vivo studies. However, it is unclear if changes are due to antipsychotic treatment. AIMS: Here we aimed to determine whether antipsychotic medication affects microglia in vivo. METHODS: To investigate this we administered two clinically relevant doses (0.05 mg n=12 and 2.5 mg n=7 slow-release pellets, placebo n=20) of haloperidol, over 2 weeks, to male Sprague Dawley rats to determine the effect on microglial cell density and morphology (area occupied by processes and microglial cell area). We developed an analysis pipeline for the automated assessment of microglial cells and used lipopolysaccharide (LPS) treatment ( n=13) as a positive control for analysis. We also investigated the effects of haloperidol ( n=9) or placebo ( n=10) on the expression of the translocator protein 18 kDa (TSPO) using autoradiography with [3H]PBR28, a TSPO ligand used in human positron emission tomography (PET) studies. RESULTS: Here we demonstrated that haloperidol at either dose does not alter microglial measures compared with placebo control animals ( p > 0.05). Similarly there was no difference in [3H]PBR28 binding between placebo and haloperidol tissue ( p > 0.05). In contrast, LPS was associated with greater cell density ( p = 0.04) and larger cell size ( p = 0.01). CONCLUSION: These findings suggest that haloperidol does not affect microglial cell density, morphology or TSPO expression, indicating that clinical study alterations are likely not the consequence of antipsychotic treatment. The automated cell evaluation pipeline was able to detect changes in microglial morphology induced by LPS and is made freely available for future use.

Brain TSPO imaging and gray matter volume in schizophrenia patients and in people at ultra high risk of psychosis: An [11C]PBR28 study.

Patients with schizophrenia show whole brain and cortical gray matter (GM) volume reductions which are progressive early in their illness. Microglia, the resident immune cells in the CNS, phagocytose neurons and synapses. Some post mortem and in vivo studies in schizophrenia show evidence for elevated microglial activation compared to matched controls. However, it is currently unclear how these results relate to changes in cortical structure. METHODS: Fourteen patients with schizophrenia and 14 ultra high risk for psychosis (UHR) subjects alongside two groups of age and genotype matched healthy controls received [11C]PBR28 PET scans to index TSPO expression, a marker of microglial activation and a 3T MRI scan. We investigated the relationship between the volume changes of cortical regions and microglial activation in cortical GM (as indexed by [11C]PBR28 distribution volume ratio (DVR). RESULTS: The total cortical GM volume was significantly lower in SCZ than the controls [mean (SD)/cm3: SCZ=448.83 (39.2) and controls=499.6 (59.2) (p=0.02) but not in UHR (mean (SD)=503.06 (57.9) and controls=524.46 (45.3) p=0.3). Regression model fitted the total cortical GM DVR values with the cortical regional volumes in SCZ (r=0.81; p<0.001) and in UHR (r=0.63; p=0.02). We found a significant negative correlation between the TSPO signal and total cortical GM volume in SCZ with the highest absolute correlation coefficient in the right superior-parietal cortex (r=-0.72; p=0.006). CONCLUSIONS: These findings suggest that microglial activity is related to the altered cortical volume seen in schizophrenia. Longitudinal investigations are required to determine whether microglial activation leads to cortical gray matter loss.

Kinetic modelling of [11C]PBR28 for 18 kDa translocator protein PET data: A validation study of vascular modelling in the brain using XBD173 and tissue analysis.

The 18 kDa translocator protein (TSPO) is a marker of microglia activation in the central nervous system and represents the main target of radiotracers for the in vivo quantification of neuroinflammation with positron emission tomography (PET). TSPO PET is methodologically challenging given the heterogeneous distribution of TSPO in blood and brain. Our previous studies with the TSPO tracers [11C]PBR28 and [11C]PK11195 demonstrated that a model accounting for TSPO binding to the endothelium improves the quantification of PET data. Here, we performed a validation of the kinetic model with the additional endothelial compartment through a displacement study. Seven subjects with schizophrenia, all high-affinity binders, underwent two [11C]PBR28 PET scans before and after oral administration of 90 mg of the TSPO ligand XBD173. The addition of the endothelial component provided a signal compartmentalization much more consistent with the underlying biology, as only in this model, the blocking study produced the expected reduction in the tracer concentration of the specific tissue compartment, whereas the non-displaceable compartment remained unchanged. In addition, we also studied TSPO expression in vessels using 3D reconstructions of histological data of frontal lobe and cerebellum, demonstrating that TSPO positive vessels account for 30% of the vascular volume in cortical and white matter.

Stress and neuroinflammation: a systematic review of the effects of stress on microglia and the implications for mental illness.

RATIONALE: Psychosocial stressors are a well-documented risk factor for mental illness. Neuroinflammation, in particular elevated microglial activity, has been proposed to mediate this association. A number of preclinical studies have investigated the effect of stress on microglial activity. However, these have not been systematically reviewed before. OBJECTIVES: This study aims to systematically review the effects of stress on microglia, as indexed by the histological microglial marker ionised calcium binding adaptor molecule 1 (Iba-1), and consider the implications of these for the role of stress in the development of mental disorders. METHODS: A systematic review was undertaken using pre-defined search criteria on PubMed and EMBASE. Inclusion and data extraction was agreed by two independent researchers after review of abstracts and full text. RESULTS: Eighteen studies met the inclusion criteria. These used seven different psychosocial stressors, including chronic restraint, social isolation and repeated social defeat in gerbils, mice and/or rats. The hippocampus (11/18 studies) and prefrontal cortex (13/18 studies) were the most frequently studied areas. Within the hippocampus, increased Iba-1 levels of between 20 and 200 % were reported by all 11 studies; however, one study found this to be a duration-dependent effect. Of those examining the prefrontal cortex, ∼75 % found psychosocial stress resulted in elevated Iba-1 activity. Elevations were also consistently seen in the nucleus accumbens, and under some stress conditions in the amygdala and paraventricular nucleus. CONCLUSIONS: There is consistent evidence that a range of psychosocial stressors lead to elevated microglial activity in the hippocampus and good evidence that this is also the case in other brain regions. These effects were seen with early-life/prenatal stress, as well as stressors in adulthood. We consider these findings in terms of the two-hit hypothesis, which proposes that early-life stress primes microglia, leading to a potentiated response to subsequent stress. The implications for understanding the pathoaetiology of mental disorders and the development of new treatments are also considered.

Microglial Activity in People at Ultra High Risk of Psychosis and in Schizophrenia: An [(11)C]PBR28 PET Brain Imaging Study.

OBJECTIVE: The purpose of this study was to determine whether microglial activity, measured using translocator-protein positron emission tomography (PET) imaging, is increased in unmedicated persons presenting with subclinical symptoms indicating that they are at ultra high risk of psychosis and to determine whether microglial activity is elevated in schizophrenia after controlling for a translocator-specific genetic polymorphism. METHOD: The authors used the second-generation radioligand [(11)C]PBR28 and PET to image microglial activity in the brains of participants at ultra high risk for psychosis. Participants were recruited from early intervention centers. The authors also imaged a cohort of patients with schizophrenia and matched healthy subjects for comparison. In total, 50 individuals completed the study. At screening, participants were genotyped to account for the rs6971 polymorphism in the gene encoding the 18Kd translocator protein. The main outcome measure was total gray matter [(11)C]PBR28 binding ratio, representing microglial activity. RESULTS: [(11)C]PBR28 binding ratio in gray matter was elevated in ultra-high-risk participants compared with matched comparison subjects (Cohen's d >1.2) and was positively correlated with symptom severity (r=0.730). Patients with schizophrenia also demonstrated elevated microglial activity relative to matched comparison subjects (Cohen's d >1.7). CONCLUSIONS: Microglial activity is elevated in patients with schizophrenia and in persons with subclinical symptoms who are at ultra high risk of psychosis and is related to at-risk symptom severity. These findings suggest that neuroinflammation is linked to the risk of psychosis and related disorders, as well as the expression of subclinical symptoms.

The methodology of TSPO imaging with positron emission tomography.

The 18-kDA translocator protein (TSPO) is consistently elevated in activated microglia of the central nervous system (CNS) in response to a variety of insults as well as neurodegenerative and psychiatric conditions. It is therefore a target of interest for molecular strategies aimed at imaging neuroinflammation in vivo. For more than 20 years, positron emission tomography (PET) has allowed the imaging of TSPO density in brain using [(11)C]-(R)-PK11195, a radiolabelled-specific antagonist of the TSPO that has demonstrated microglial activation in a large number pathological cohorts. The significant clinical interest in brain immunity as a primary or comorbid factor in illness has sparked great interest in the TSPO as a biomarker and a surprising number of second generation TSPO radiotracers have been developed aimed at improving the quality of TSPO imaging through novel radioligands with higher affinity. However, such major investment has not yet resulted in the expected improvement in image quality. We here review the main methodological aspects of TSPO PET imaging with particular attention to TSPO genetics, cellular heterogeneity of TSPO in brain tissue and TSPO distribution in blood and plasma that need to be considered in the quantification of PET data to avoid spurious results as well as ineffective development and use of these radiotracers.

Neurological deficits caused by tissue hypoxia in neuroinflammatory disease.

OBJECTIVE: To explore the presence and consequences of tissue hypoxia in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). METHODS: EAE was induced in Dark Agouti rats by immunization with recombinant myelin oligodendrocyte glycoprotein and adjuvant. Tissue hypoxia was assessed in vivo using 2 independent methods: an immunohistochemical probe administered intravenously, and insertion of a physical, oxygen-sensitive probe into the spinal cord. Indirect markers of tissue hypoxia (eg, expression of hypoxia-inducible factor-1α [HIF-1α], vessel diameter, and number of vessels) were also assessed. The effects of brief (1 hour) and continued (7 days) normobaric oxygen treatment on function were evaluated in conjunction with other treatments, namely administration of a mitochondrially targeted antioxidant (MitoQ) and inhibition of inducible nitric oxide synthase (1400W). RESULTS: Observed neurological deficits were quantitatively, temporally, and spatially correlated with spinal white and gray matter hypoxia. The tissue expression of HIF-1α also correlated with loss of function. Spinal microvessels became enlarged during the hypoxic period, and their number increased at relapse. Notably, oxygen administration significantly restored function within 1 hour, with improvement persisting at least 1 week with continuous oxygen treatment. MitoQ and 1400W also caused a small but significant improvement. INTERPRETATION: We present chemical, physical, immunohistochemical, and therapeutic evidence that functional deficits caused by neuroinflammation can arise from tissue hypoxia, consistent with an energy crisis in inflamed central nervous system tissue. The neurological deficit was closely correlated with spinal white and gray matter hypoxia. This realization may indicate new avenues for therapy of neuroinflammatory diseases such as MS.