Systematic assessment of the replicability and generalizability of preclinical findings: Impact of protocol harmonization across laboratory sites.

The influence of protocol standardization between laboratories on their replicability of preclinical results has not been addressed in a systematic way. While standardization is considered good research practice as a means to control for undesired external noise (i.e., highly variable results), some reports suggest that standardized protocols may lead to idiosyncratic results, thus undermining replicability. Through the EQIPD consortium, a multi-lab collaboration between academic and industry partners, we aimed to elucidate parameters that impact the replicability of preclinical animal studies. To this end, 3 experimental protocols were implemented across 7 laboratories. The replicability of results was determined using the distance travelled in an open field after administration of pharmacological compounds known to modulate locomotor activity (MK-801, diazepam, and clozapine) in C57BL/6 mice as a worked example. The goal was to determine whether harmonization of study protocols across laboratories improves the replicability of the results and whether replicability can be further improved by systematic variation (heterogenization) of 2 environmental factors (time of testing and light intensity during testing) within laboratories. Protocols were tested in 3 consecutive stages and differed in the extent of harmonization across laboratories and standardization within laboratories: stage 1, minimally aligned across sites (local protocol); stage 2, fully aligned across sites (harmonized protocol) with and without systematic variation (standardized and heterogenized cohort); and stage 3, fully aligned across sites (standardized protocol) with a different compound. All protocols resulted in consistent treatment effects across laboratories, which were also replicated within laboratories across the different stages. Harmonization of protocols across laboratories reduced between-lab variability substantially compared to each lab using their local protocol. In contrast, the environmental factors chosen to introduce systematic variation within laboratories did not affect the behavioral outcome. Therefore, heterogenization did not reduce between-lab variability further compared to the harmonization of the standardized protocol. Altogether, these findings demonstrate that subtle variations between lab-specific study protocols may introduce variation across independent replicate studies even after protocol harmonization and that systematic heterogenization of environmental factors may not be sufficient to account for such between-lab variation. Differences in replicability of results within and between laboratories highlight the ubiquity of study-specific variation due to between-lab variability, the importance of transparent and fine-grained reporting of methodologies and research protocols, and the importance of independent study replication.

[18F]MPPF and [18F]FDG µPET imaging in rats: impact of transport and restraint stress.

BACKGROUND: Stress exposure can significantly affect serotonergic signaling with a particular impact on 5-HT1A receptor expression. Positron emission tomography (PET) provides opportunities for molecular imaging of alterations in 5-HT1A receptor binding following stress exposure. Considering the possible role of 5-HT1A receptors in stress coping mechanisms, respective imaging approaches are of particular interest. MATERIAL AND METHODS: For twelve consecutive days, Sprague Dawley rats were exposed to daily transport with a 1 h stay in a laboratory or daily transport plus 1 h restraint in a narrow tube. Following, animals were subjected to µPET imaging with 2'-methoxyphenyl-(N-2'-pyridinyl)-p-[18F]fluoro-benzamidoethylpiperazine ([18F]MPPF) and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). Behavioral and biochemical parameters were analyzed to obtain additional information. RESULTS: In rats with repeated transport, hippocampal [18F]MPPF binding exceeded that in the naive group, while no difference in [18F]FDG uptake was detected between the groups. A transient decline in body weight was observed in rats with transport or combined transport and restraint. Thereby, body weight development correlated with [18F]MPPF binding. CONCLUSIONS: Mild-to-moderate stress associated with daily transport and exposure to a laboratory environment can trigger significant alterations in hippocampal binding of the 5-HT1A receptor ligand [18F]MPPF. This finding indicates that utmost care is necessary to control and report transport and associated handling procedures for animals used in µPET studies analyzing the serotonergic system in order to enhance the robustness of conclusions and allow replicability of findings. In view of earlier studies indicating that an increase in hippocampal 5-HT1A receptor expression may be associated with a resilience to stress, it would be of interest to further evaluate 5-HT1A receptor imaging approaches as a candidate biomarker for the vulnerability to stress.

Systematic review of guidelines for internal validity in the design, conduct and analysis of preclinical biomedical experiments involving laboratory animals.

Over the last two decades, awareness of the negative repercussions of flaws in the planning, conduct and reporting of preclinical research involving experimental animals has been growing. Several initiatives have set out to increase transparency and internal validity of preclinical studies, mostly publishing expert consensus and experience. While many of the points raised in these various guidelines are identical or similar, they differ in detail and rigour. Most of them focus on reporting, only few of them cover the planning and conduct of studies. The aim of this systematic review is to identify existing experimental design, conduct, analysis and reporting guidelines relating to preclinical animal research. A systematic search in PubMed, Embase and Web of Science retrieved 13 863 unique results. After screening these on title and abstract, 613 papers entered the full-text assessment stage, from which 60 papers were retained. From these, we extracted unique 58 recommendations on the planning, conduct and reporting of preclinical animal studies. Sample size calculations, adequate statistical methods, concealed and randomised allocation of animals to treatment, blinded outcome assessment and recording of animal flow through the experiment were recommended in more than half of the publications. While we consider these recommendations to be valuable, there is a striking lack of experimental evidence on their importance and relative effect on experiments and effect sizes.

Imaging biomarkers of behavioral impairments: A pilot micro-positron emission tomographic study in a rat electrical post-status epilepticus model.

OBJECTIVE: In patients with epilepsy, psychiatric comorbidities can significantly affect the disease course and quality of life. Detecting and recognizing these comorbidities is central in determining an optimal treatment plan. One promising tool in detecting biomarkers for psychiatric comorbidities in epilepsy is positron emission tomography (PET). METHODS: Behavioral and biochemical variables were cross-correlated with the results from two µPET scans using the tracers [18 F]fluoro-2-deoxy-D-glucose ([18 F]FDG) and 2'-methoxyphenyl-(N-2'-pyridinyl)-p-18 F-fluoro-benzamidoethylpiperazine ([18 F]MPPF) to explore potential biomarkers for neurobehavioral comorbidities in an electrically induced post-status epilepticus rat model of epilepsy. RESULTS: In rats with epilepsy, µPET analysis revealed a local reduction in hippocampal [18 F]FDG uptake, and a local increase in [18 F]MPPF binding. These changes exhibited a correlation with burrowing as a "luxury" behavior, social interaction, and anxiety-associated behavioral patterns. Interestingly, hippocampal [18 F]FDG uptake did not correlate with spontaneous recurrent seizure activity. SIGNIFICANCE: In the electrically induced post-status epilepticus rat model, we demonstrated hippocampal hypometabolism and its correlation with a range of neurobehavioral alterations. These findings require further confirmation in other preclinical models and patients with epilepsy and psychiatric disorders to address the value of [18 F]FDG uptake as an imaging biomarker candidate for psychiatric comorbidities in patients as well as for severity assessment in rodent epilepsy models.

Imaging correlates of behavioral impairments: An experimental PET study in the rat pilocarpine epilepsy model.

Psychiatric comorbidities are prevalent in patients with epilepsy and greatly contribute to the overall burden of disease. The availability of reliable biomarkers to diagnose epilepsy-associated comorbidities would allow for effective treatment and improved disease management. Due to their non-invasive nature, molecular imaging techniques such as positron emission tomography (PET) are ideal tools to measure pathologic changes. In the current study we investigated the potential of [18F]fluoro-2-deoxy-d-glucose ([18F]FDG) and 2'-methoxyphenyl-(N-2'-pyridinyl)-p-18F-fluoro-benzamidoethylpiperazine ([18F]MPPF) as imaging correlates of neurobehavioral comorbidities in the pilocarpine rat model of epilepsy. Findings from rats with epilepsy revealed a regional reduction in [18F]FDG uptake indicating thalamic hypometabolism. In addition, an increase in septal [18F]MPPF binding was observed in rats with spontaneous recurrent seizures. Both thalamic [18F]FDG and septal [18F]MPPF data proved to correlate with behavioral alterations including decreases in luxury behavior such as burrowing and social interaction, and changes in behavioral patterns in anxiety tests. A correlation with seizure frequency was confirmed for thalamic [18F]FDG data. Moreover, thalamic [18F]FDG and septal [18F]MPPF data exhibited a correlation with brain-derived neurotrophic factor (BDNF) serum concentrations, which were lowered in rats with epilepsy. In conclusion, µPET data from rats with pilocarpine-induced epileptogenesis indicate altered septal 5-HT1A receptor binding. Further research is necessary assessing whether septal 5-HT1A receptor binding may serve as an imaging correlate of neuropsychiatric comorbidities in epilepsy patients and for severity assessment in rodent epilepsy models. In contrast, we obtained evidence that [18F]FDG uptake also reflects the severity of epilepsy and, thus, might not constitute a biomarker with sufficient specificity for psychiatric comorbidities. Evidence has been obtained that BDNF might serve as a peripheral circulatory biomarker. Further experimental and clinical assessment is necessary for validation of the marker candidates.

Evaluation of Small-Animal PET Outcome Measures to Detect Disease Modification Induced by BACE Inhibition in a Transgenic Mouse Model of Alzheimer Disease.

In this study, we investigated the effects of chronic administration of an inhibitor of the ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) on Alzheimer-related pathology by multitracer PET imaging in transgenic APPPS1-21 (TG) mice. Methods: Wild-type (WT) and TG mice received vehicle or BACE inhibitor (60 mg/kg) starting at 7 wk of age. Outcome measures of brain metabolism, neuroinflammation, and amyloid-ß pathology were obtained through small-animal PET imaging with 18F-FDG, 18F-peripheral benzodiazepine receptor (18F-PBR), and 18F-florbetapir (18F-AV45), respectively. Baseline scans were acquired at 6-7 wk of age and follow-up scans at 4, 7, and 12 mo. 18F-AV45 uptake was measured at 8 and 13 mo of age. After the final scans, histologic measures of amyloid-ß (4G8), microglia (ionized calcium binding adaptor molecule 1), astrocytes (glial fibrillary acidic protein), and neuronal nuclei were performed. Results: TG mice demonstrated significant age-associated increases in 18F-AV45 uptake. An effect of treatment was observed in the cortex (P = 0.0014), hippocampus (P = 0.0005), and thalamus (P < 0.0001). Histology confirmed reduction of amyloid-ß pathology in TG-BACE mice. Regardless of treatment, TG mice demonstrated significantly lower 18F-FDG uptake than WT mice in the thalamus (P = 0.0004) and hippocampus (P = 0.0332). Neuronal nucleus staining was lower in both TG groups in the thalamus and cortex. 18F-PBR111 detected a significant age-related increase in TG mice (P < 0.0001) but did not detect the treatment-induced reduction in activated microglia as demonstrated by histology. Conclusion: Although 18F-FDG, 18F-PBR111, and 18F-AV45 all detected pathologic alterations between TG and WT mice, only 18F-AV45 could detect an effect of BACE inhibitor treatment. However, changes in WT binding of 18F-AV45 undermine the specificity of this effect.

Longitudinal Characterization of [18F]-FDG and [18F]-AV45 Uptake in the Double Transgenic TASTPM Mouse Model.

We aimed to monitor the timing of amyloid-ß deposition in relation to changes in brain function using in vivo imaging with [18F]-AV45 and [18F]-FDG in a mouse model of Alzheimer's disease. TASTPM transgenic mice and wild-type controls were scanned longitudinally with [18F]-AV45 and [18F]-FDG before (3 months of age) and at multiple time points after the onset of amyloid deposition (6, 9, 12, and 15 months of age). As expected with increasing amyloidosis, TASTPM mice demonstrated progressive age-dependent increases in [18F]-AV45 uptake that were significantly higher than for WT from 9 months onwards and correlated to ex vivo measures of amyloid burden. The metabolism of [18F]-AV45 produces several brain penetrant radiometabolites and normalization to a reference region helps to negate this non-specific binding and improve the sensitivity of [18F]-AV45. The observed trajectory of [18F]-FDG alterations deviated from our proposed hypothesis of gradual decreases with worsening amyloidosis. While [18F]-FDG uptake in TASTPM mice was significantly lower than that of WT at 9 months, reduced [18F]-FDG was not associated with aging in TASTPM mice. Moreover, [18F]-FDG uptake did not correlate to measures of ex vivo amyloid burden. Our findings suggest that while amyloid-ß is sufficient to induce hypometabolism, these pathologies are not linked in a dose-dependent manner in TASTPM mice.

In Vivo Amyloid-ß Imaging in the APPPS1-21 Transgenic Mouse Model with a (89)Zr-Labeled Monoclonal Antibody.

INTRODUCTION: The accumulation of amyloid-ß is a pathological hallmark of Alzheimer's disease and is a target for molecular imaging probes to aid in diagnosis and disease monitoring. This study evaluated the feasibility of using a radiolabeled monoclonal anti-amyloid-ß antibody (JRF/AßN/25) to non-invasively assess amyloid-ß burden in aged transgenic mice (APPPS1-21) with µPET imaging. METHODS: We investigated the antibody JRF/AßN/25 that binds to full-length Aß. JRF/AßN/25 was radiolabeled with a [(89)Zr]-desferal chelate and intravenously injected into 12-13 month aged APPPS1-21 mice and their wild-type (WT) controls. Mice underwent in vivo µPET imaging at 2, 4, and 7 days post injection and were sacrificed at the end of each time point to assess brain penetrance, plaque labeling, biodistribution, and tracer stability. To confirm imaging specificity we also evaluated brain uptake of a non-amyloid targeting [(89)Zr]-labeled antibody (trastuzumab) as a negative control, additionally we performed a competitive blocking study with non-radiolabeled Df-Bz-JRF/AßN/25 and finally we assessed the possible confounding effects of blood retention. RESULTS: Voxel-wise analysis of µPET data demonstrated significant [(89)Zr]-Df-Bz-JRF/AßN/25 retention in APPPS1-21 mice at all time points investigated. With ex vivo measures of radioactivity, significantly higher retention of [(89)Zr]-Df-Bz-JRF/AßN/25 was found at 4 and 7 days pi in APPPS1-21 mice. Despite the observed genotypic differences, comparisons with immunohistochemistry revealed that in vivo plaque labeling was low. Furthermore, pre-treatment with Df-Bz-JRF/AßN/25 only partially blocked [(89)Zr]-Df-Bz-JRF/AßN/25 uptake indicative of a high contribution of non-specific binding. CONCLUSION: Amyloid plaques were detected in vivo with a radiolabeled monoclonal anti-amyloid antibody. The low brain penetrance of the antibody in addition to non-specific binding prevented an accurate estimation of plaque burden. However, it should be noted that [(89)Zr]-Df-Bz-JRF/AßN/25 nevertheless demonstrated in vivo binding and strategies to increase brain penetrance would likely achieve better results.

The Effects of Physiological and Methodological Determinants on 18F-FDG Mouse Brain Imaging Exemplified in a Double Transgenic Alzheimer Model.

INTRODUCTION: In this study, the influence of physiological determinants on 18F-fluoro-d-glucose ((18)F-FDG) brain uptake was evaluated in a mouse model of Alzheimer disease. MATERIALS AND METHODS: TASTPM (Tg) and age-matched C57BL/6 J (WT) mice were fasted for 10 hours, while another group was fasted for 20 hours to evaluate the effect of fasting duration. The effect of repeatedly scanning was evaluated by scanning Tg and WT mice at days 1, 4, and 7. Brain (18)F-FDG uptake was evaluated in the thalamus being the most indicative region. Finally, the cerebellum was tested as a reference region for the relative standard uptake value (rSUV). RESULTS: When correcting the brain uptake for glucose, the effect of different fasting durations was attenuated and the anticipated hypometabolism in Tg mice was demonstrated. Also, with repeated scanning, the brain uptake values within a group and the hypometabolism of the Tg mice only remained stable over time when glucose correction was applied. Finally, hypometabolism was also observed in the cerebellum, yielding artificially higher rSUV values for Tg mice. CONCLUSION: Corrections for blood glucose levels have to be applied when semiquantifying (18)F-FDG brain uptake in mouse models for AD. Potential reference regions for normalization should be thoroughly investigated to ensure that they are not pathologically affected also by afferent connections.

Synthesis and Evaluation of a Zr-89-Labeled Monoclonal Antibody for Immuno-PET Imaging of Amyloid-ß Deposition in the Brain.

PURPOSE: The aim of this study was to evaluate the in vitro and in vivo characteristics of [(89)Zr]JRF/AßN/25, a radiolabeled monoclonal antibody directed against amyloid-ß (Aß). PROCEDURES: JRF/AßN/25 was labeled with (89)Zr following modification with desferal. The affinity of the tracer for Aß1-40 was determined in a saturation binding assay. In vitro stability was evaluated, and in vivo plasma stability and biodistribution of [(89)Zr]Df-Bz-JRF/AßN/25 were determined in wild-type mice. To evaluate whether the antibody can cross the blood-brain barrier, brain uptake in wild-type mice was additionally assessed by ex vivo autoradiography. RESULTS: [(89)Zr]Df-Bz-JRF/AßN/25 was obtained in an average radiochemical yield of 50 % and a radiochemical purity of >97 %. A saturation binding assay demonstrated specific binding of [(89)Zr]Df-Bz-JRF/AßN/25 to Aß1-40 with nanomolar affinity. The tracer was stable in buffer and proved to be stable in vivo with >92 % intact monoclonal antibody (mAb) remaining in the plasma at 48 h post injection. A biodistribution study showed a slow blood clearance with no significant accumulation of activity in any of the organs. Furthermore, [(89)Zr]Df-Bz-JRF/AßN/25 demonstrated modest brain penetration, which slowly decreased in time. This cerebral uptake was confirmed by ex vivo autoradiography. CONCLUSIONS: [(89)Zr]Df-Bz-JRF/AßN/25 binds with high affinity to Aß1-40. The tracer displays an acceptable in vivo stability and is able to cross the blood-brain barrier. [(89)Zr]Df-Bz-JRF/AßN/25 might therefore be a potential candidate for in vivo imaging of Aß deposition in the brain.

In vivo molecular neuroimaging of glucose utilization and its association with fibrillar amyloid-ß load in aged APPPS1-21 mice.

INTRODUCTION: Radioligand imaging is a powerful in vivo method to assess the molecular basis of Alzheimer's Disease. We therefore aimed to visualize the pathological deposition of fibrillar amyloid-ß and neuronal dysfunction in aged double transgenic mice. METHODS: Using non-invasive positron emission tomography (PET) we assessed brain glucose utilization with [(18)F]FDG and fibrillar amyloidosis with [(11)C]PiB and [(18)F]AV45 in 12 month old APPPS1-21 (n = 10) mice and their age-matched wild-type controls (n = 15). PET scans were analyzed with statistical parametric mapping (SPM) to detect significant differences in tracer uptake between genotypes. After imaging, mice were sacrificed and ex vivo measures of amyloid-ß burden with immunohistochemistry as well as glucose utilization with [(14)C]-2DG autoradiography were obtained as gold standards. RESULTS: Voxel-wise SPM analysis revealed significantly decreased [(18)F]FDG uptake in aged APPPS1-21 mice in comparison to WT with the thalamus (96.96 %, maxT = 3.35) and striatum (61.21 %, maxT = 3.29) demonstrating the most widespread reductions at the threshold of p < 0.01. [(11)C]PiB binding was significantly increased in APPPS1-21 mice, most notably in the hippocampus (87.84 %, maxT = 7.15) and cortex (69.08 %, maxT = 7.95), as detected by SPM voxel-wise analysis at the threshold of p < 0.01. Using the same threshold [(18)F]AV45 uptake was comparably lower with less significant differences. Compared to their respective ex vivo equivalents [(18)F]FDG demonstrated significant positive correlation to [(14)C]2-DG autoradiography (r = 0.67, p <0.0001) while [(11)C]PiB and [(18)F]AV45 binding did not correlate to ex vivo immunohistochemistry for amyloid-ß (r = 0.25, p = 0.07 and r = 0.17, p = 0.26 respectively). Lastly no correlation was observed between regions of high amyloid burden and those with decreased glucose utilization (r = 0.001, p = 0.99). CONCLUSIONS: Our findings support that fibrillar amyloid-ß deposition and reduced glucose utilization can be visualized and quantified with in vivo µPET imaging in aged APPPS1-21 mice. Therefore, the combined use of [(18)F]FDG and amyloid µPET imaging can shed light on the underlying relationship between fibrillar amyloid-ß pathology and neuronal dysfunction.

Quantitative µPET Imaging of Cerebral Glucose Metabolism and Amyloidosis in the TASTPM Double Transgenic Mouse Model of Alzheimer's Disease.

Positron emission tomography studies of cerebral glucose utilization and amyloid-ß deposition with fluoro-deoxy-D-glucose ([(18)F]-FDG) and amyloid tracers have shown characteristic pathological changes in Alzheimer's Disease that can be used for disease diagnosis and monitoring. Application of this technology to preclinical research with transgenic animal models would greatly facilitate drug discovery and further understanding of disease processes. The results from preclinical studies with these imaging biomarkers have however been highly inconsistent, causing doubts over whether animal models can truly replicate an AD-like phenotype. In this study we performed in vivo imaging with [(18)F]-FDG and [(18)F]-AV45 in double transgenic TASTPM mice, a transgenic model that been previously demonstrated high levels of fibrillar amyloid-ß and decreases in cerebral glucose utilization with ex vivo techniques. Our results show widespread and significant retention of [(18)F]-AV45 (p < 0.0001) in aged TASTPM mice in addition to significant regional decreases in [(18)F]-FDG uptake (p < 0.05). In vivo quantification of amyloid-ß showed a strong (Pearson's r = 0.7078), but not significant (p = 0.1156), positive correlation with ex vivo measures suggesting some limitations on tracer sensitivity. In the case of [(18)F]-FDG, voxelwise analysis greatly enhanced detection of hypometabolic regions. We further evidenced modest neuronal loss (thalamus p = 0.0318) that could underlie the observed hypometabolism. This research was performed in conjunction with the European Community's Seventh Framework Program (FP7/2007-2013) for the Innovative Medicine Initiative under the PharmaCog Grant Agreement no.115009.

Preclinical Comparison of the Amyloid-ß Radioligands [(11)C]Pittsburgh compound B and [(18)F]florbetaben in Aged APPPS1-21 and BRI1-42 Mouse Models of Cerebral Amyloidosis.

PURPOSE: The aim of this study was to compare [(11)C]Pittsburgh compound B ([(11)C]PiB) and [(18)F]florbetaben ([(18)F]FBB) for preclinical investigations of amyloid-ß pathology. PROCEDURES: We investigated two aged animal models of cerebral amyloidosis with contrasting levels of amyloid-ß relating to "high" (APPPS1-21 n = 6, wild type (WT) n = 7) and "low" (BRI1-42 n = 6, WT n = 6) target states, respectively. RESULTS: APPPS1-21 mice (high target state) demonstrated extensive fibrillar amyloid-ß deposition that translated to significantly increased retention of [(11)C]PiB and [(18)F]FBB in comparison to their wild type. The retention pattern of [(11)C]PiB and [(18)F]FBB in this cohort displayed a significant correlation. However, the relative difference in tracer uptake between diseased and healthy mice was substantially higher for [(11)C]PiB than for [(18)F]FBB. Although immunohistochemistry confirmed the high plaque load in APPPS1-21 mice, correlation between tracer uptake and ex vivo quantification of amyloid-ß was poor for both tracers. BRI1-42 mice (low target state) did not demonstrate increased tracer uptake. CONCLUSIONS: In cases of high fibrillar amyloid-ß burden, both tracers detected significant differences between diseased and healthy mice, with [(11)C]PiB showing a larger dynamic range.

In vivo evaluation of (18)F-labeled TCO for pre-targeted PET imaging in the brain.

INTRODUCTION: The tetrazine-trans-cylooctene cycloaddition using radiolabeled tetrazine or radiolabeled trans-cyclooctene (TCO) has been reported to be a very fast, selective and bioorthogonal reaction that could be useful for in vivo radiolabeling of molecules. We wanted to evaluate the in vivo biodistribution profile and brain uptake of (18)F-labeled TCO ([(18)F]TCO) to assess its potential for pre-targeted imaging in the brain. METHODS: We evaluated the in vivo behavior of [(18)F]TCO via an ex vivo biodistribution study complemented by in vivo µPET imaging at 5, 30, 60, 90, 120 and 240 min post tracer injection. An in vivo metabolite study was performed at 5 min, 30 min and 120 min post [(18)F]TCO injection by RP-HPLC analysis of plasma and brain extracts. Incubation with human liver microsomes was performed to further evaluate the metabolite profile of the tracer. RESULTS: µPET imaging and ex-vivo biodistribution revealed an high initial brain uptake of [(18)F]TCO (3.8%ID/g at 5 min pi) followed by a washout to 3.0%ID/g at 30 min pi. Subsequently the brain uptake increased again to 3.7%ID/g at 120 min pi followed by a slow washout until 240 min pi (2.9%ID/g). Autoradiography confirmed homogenous brain uptake. On the µPET images bone uptake became gradually visible after 120 min pi and was clearly visible at 240 min pi. The metabolite study revealed a fast metabolization of [(18)F]TCO in plasma and brain into three main polar radiometabolites. CONCLUSIONS: Although [(18)F]TCO has previously been described to be a useful tracer for radiolabeling of tetrazine modified targeting molecules, our study indicates that its utility for in vivo chemistry and pre-targeted imaging will be limited. Although [(18)F]TCO clearly enters the brain, it is quickly metabolized with a non-specific accumulation of radioactivity in the brain and bone.

Inhibition of stress-induced hepatic tryptophan 2,3-dioxygenase exhibits antidepressant activity in an animal model of depressive behaviour.

The role of hepatic tryptophan 2,3 dioxygenase (TDO) was assessed in the provocation of stress-induced depression-related behaviour in the rat. TDO drives tryptophan metabolism via the kynurenine pathway (KP) and leads to the production of neuroactive metabolites including kynurenine. A single 2 h period of restraint stress in adult male Sprague-Dawley rats provoked an increase in circulating concentrations of the glucocorticoid corticosterone and induction of hepatic TDO expression and activity. Repeated exposure to stress (10 d of 2 h restraint each day) provoked an increase in immobility in the forced swimming test (FST) indicative of depression-related behaviour. Immobility was accompanied by an increase in the circulating corticosterone concentrations, expression and activity of hepatic TDO and increase in the expression of TDO in the cerebral cortex. Increased TDO activity was associated with raised circulating kynurenine concentrations and a reduction in circulating tryptophan concentrations indicative of KP activation. Co-treatment with the TDO inhibitor allopurinol (20 mg/kg, i.p.), attenuated the chronic stress-related increase in immobility in the FST and the accompanying increase in circulating kynurenine concentrations. These findings indicate that stress-induced corticosterone and consequent activation of hepatic TDO, tryptophan metabolism and production of kynurenine provoke a depression-related behavioural phenotype. Inhibition of stress-related hepatic TDO activity promotes antidepressant activity. TDO may therefore represent a promising target for the treatment of depression associated with stress-related disorders in which there is evidence for KP activation.