Jeroen Verhaeghe

Brain inflammation in a chronic epilepsy model: Evolving pattern of the translocator protein during epileptogenesis.

AIMS A hallmark in the neuropathology of temporal lobe epilepsy is brain inflammation which has been suggested as both a biomarker and a new mechanistic target for treatments. The translocator protein (TSPO), due to its high upregulation under neuroinflammatory conditions and the availability of selective PET tracers, is a candidate target. An important step to exploit this target is a thorough characterisation of the spatiotemporal profile of TSPO during epileptogenesis. METHODS TSPO expression, microglial activation, astrocyte reactivity and cell loss in several brain regions were evaluated at five time points during epileptogenesis, including the chronic epilepsy phase in the kainic acid-induced status epilepticus (KASE) model (n = 52) and control Wistar Han rats (n = 33). Seizure burden was also determined in the chronic phase. Furthermore, ¹⁸F-PBR111 PET/MRI scans were acquired longitudinally in an additional four KASE animals. RESULTS TSPO expression measured with in vitro and in vivo techniques was significantly increased at each time point and peaked two weeks post-SE in the limbic system. A prominent association between TSPO expression and activated microglia (p < 0.001; r = 0.7), as well as cell loss (p < 0.001; r = -0.8) could be demonstrated. There was a significant positive correlation between spontaneous seizures and TSPO upregulation in several brain regions with increased TSPO expression. CONCLUSIONS TSPO expression was dynamically upregulated during epileptogenesis, persisted in the chronic phase and correlated with microglia activation rather than reactive astrocytes. TSPO expression was correlating with spontaneous seizures and its high expression during the latent phase might possibly suggest being an important switching point in disease ontogenesis which could be further investigated by PET imaging.

Towards a reproducible protocol for repetitive and semi-quantitative rat brain imaging with (18) F-FDG: exemplified in a memantine pharmacological challenge.

The standard uptake value (SUV), commonly used to quantify (18)F-FluoroDeoxyGlucose (FDG) uptake in small animal brain PET imaging, is affected by many factors. In this study the influence of fasting times, inter-scan duration and repetitive scanning on the variability of different SUV measures is investigated. Additionally it is demonstrated that these variables could adversely influence the outcome of a pharmacological challenge when not accounted for. Naive Sprague-Dawley rats (n=20) were randomly divided into five different fasting groups (no fasting up to 24h of fasting). SUV brain uptake values were reproducible in naive animals when a fasting period of at least 12h is used and for shorter fasting periods SUV values need to be corrected for the glucose level. Additionally, a separate animal group (n=6) was sufficiently fasted for 16h and in a longitudinal setting being scanned six times in three weeks. Especially with short inter-scan durations, increasing glucose levels were found over time which was attributed to increased stress due to repeated food deprivation, altered food intake or scan manipulations. As a result, even with controlled and sufficient fasting, blood glucose levels should be taken into account for data quantification. Strikingly, even the brain activation effects of an NMDA-antagonist challenge with memantine could not be detected in experiments with a short inter-scan duration if glucose levels were not taken into account. Correcting for glucose levels decreases the inter- and intra-animal variability for rat brain imaging. SUV corrected for glucose levels yields the lowest inter-animal variation. However, if the body weight changes significantly, as in a long experiment, quantification based on the glucose corrected percentage injected dose (and not SUV) is recommendable as this yields the lowest intra-animal variation.

N-Acetylcysteine– and MK-801–Induced Changes in Glutamate Levels Do Not Affect In Vivo Binding of Metabotropic Glutamate 5 Receptor Radioligand 11C-ABP688 in Rat Brain

Abnormal glutamate transmission is involved in various neurologic disorders, such as epilepsy, schizophrenia, and Parkinson disease. At present, no imaging techniques are capable of measuring acute fluctuations in endogenous glutamate levels in vivo. We evaluated the potential of 11C-ABP688, a PET ligand that binds to an allosteric site of the metabotropic glutamate 5 receptor, in rats by using small-animal PET and β-microprobes after pharmacologic challenges with N-acetylcysteine (NAc) and MK-801. Both compounds are known to induce increases in endogenous glutamate levels. Methods: Three experiments with 11C-ABP688 were performed to validate our study setup: first, metabolite analyses during workup (n = 3) and after a selected treatment (n = 3); second, a test–retest (n = 12) small-animal PET experiment (1 h scan; 27.75 MBq of 11C-ABP688 administered intravenously; <3 nmol/kg); and third, a small-animal PET and β-microprobe cold blocking study (n = 6/condition) with unlabeled ABP688. After this experimental validation, rats were pretreated with either NAc (intravenous infusion of 50 mg/kg/h) or MK-801 (0.16 mg/kg; given intraperitoneally); this step was followed by small-animal PET with 11C-ABP688 (n = 12) or β-microprobe measurements (n = 10/condition) of 11C-ABP688. Time–activity curves were extracted, and the nondisplaceable binding potential (BPND) was calculated by use of the simplified reference tissue model with the cerebellum as a reference region. Results: 11C-ABP688 BPND measurements were highly reproducible (test–retest), and both small-animal PET and β-microprobes were able to discriminate changes in 11C-ABP688 binding (cold blocking). The average small-animal PET BPND measurements in the test experiment for the caudate putamen, frontal cortex, cerebral cortex, hippocampus, and thalamus were 2.58, 1.40, 1.60, 1.86, and 1.09, respectively. However, no significant differences in BPND measurements were observed with small-animal PET in the test and retest conditions on the one hand and the NAc and MK-801 conditions on the other hand for any of these regions. When β-microprobes were used, the average BPND in the caudate putamen was 0.94, and no significant changes in the test and MK-801 conditions were observed. Conclusion: Pharmacologic challenges with NAc and MK-801 did not affect the 11C-ABP688 BPND in the rat brain. These data suggest that the in vivo affinity of 11C-ABP688 for binding to an allosteric site of the metabotropic glutamate 5 receptor is not modulated by changes in glutamate levels and that 11C-ABP688 is not capable of measuring acute fluctuations in endogenous levels of glutamate in vivo in the rat brain.

Small-animal repetitive transcranial magnetic stimulation combined with [18F]-FDG microPET to quantify the neuromodulation effect in the rat brain

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neurostimulation technique for the treatment of various neurological and psychiatric disorders. To investigate the working mechanism of this treatment approach, we designed a small-animal coil for dedicated use in rats and we combined this neurostimulation method with small-animal positron emission tomography (microPET or μPET) to quantify regional 2-deoxy-2-((18)F)fluoro-d-glucose ([(18)F]-FDG) uptake in the rat brain, elicited by a low- (1 Hz) and a high- (50 Hz) frequency paradigm. Rats (n=6) were injected with 1 mCi of [(18)F]-FDG 10 min after the start of 30 min of stimulation (1 Hz, 50 Hz or sham), followed by a 20-min μPET image acquisition. Voxel-based statistical parametric mapping (SPM) image analysis of 1-Hz and 50-Hz versus sham stimulation was performed. For both the 1-Hz and 50-Hz paradigms we found a large [(18)F]-FDG hypermetabolic cluster (2.208 mm(3) and 2.616 mm(3), resp.) (analysis of variance (ANOVA), p<0.05) located in the dentate gyrus complemented with an additional [(18)F]-FDG hypermetabolic cluster (ANOVA, p<0.05) located in the entorhinal cortex (2.216 mm(3)) for the 50-Hz stimulation. The effect on [(18)F]-FDG metabolism was 2.9 ± 0.8% at 1 Hz and 2.5 ± 0.8% at 50 Hz for the dentate gyrus clusters and 3.3 ± 0.5% for the additional cluster in the entorhinal cortex at 50 Hz. The maximal (4.19 vs. 2.58) and averaged (2.87 vs. 2.21) T-values are higher for 50 Hz versus 1 Hz. This experimental study demonstrates the feasibility to combine μPET imaging in rats stimulated with rTMS using a custom-made small-animal magnetic stimulation setup to quantify changes in the cerebral [(18)F]-FDG uptake as a measure for neuronal activity.

Simultaneous water activation and glucose metabolic rate imaging with PET.

Simultaneous imaging of [15O]H2O activation measuring relative regional cerebral blood flow (rrCBF) and [18F]FDG for glucose metabolism is of potential interest for presurgical positron emission tomography (PET) imaging of brain tumours. A novel imaging strategy is proposed to be able to separate the [18F]FDG and the multiple [15O]H2O signals from a simultaneously acquired dynamic acquisition of the two PET tracers. The technique is based on the fact that the dynamics of the two tracers are very distinct. By adopting an appropriate bolus injection strategy and by defining tailored sets of basis functions that model either the FDG or water component it is possible to separate the FDG and water signal. The basis functions are defined as the convolution of estimated input functions (IPFs) with a set of decaying exponential functions, where the IPFs are estimated from the overall measured head curve. The estimated IPFs are not required to correspond to the arterial IPFs of the two components and the proposed method does not need an arterial blood input function, which greatly increases the practical feasibility of the method. Once the IPFs are estimated the voxel-level time activity curves (TACs) are modelled using the basis functions. The set of exponential functions used to define the FDG basis functions are predetermined and equal for all voxels while the set of exponential functions defining the water basis functions will be determined for each individual voxel. The technique can be applied post-reconstruction as a fitting routine using the MLEM algorithm, or, the model can be incorporated in a global 4D reconstruction strategy to further reduce the noise in the estimated components. In this work the technique is applied post-reconstruction. Simulation studies show that the [18F]FDG and the multiple [15O]H2O components can be separated, when using simultaneous imaging, by the proposed model. The resulting errors are similar to the errors obtained when the two tracers are imaged separately demonstrating the feasibility of the method.

Non-invasive PET imaging of brain inflammation at disease onset predicts spontaneous recurrent seizures and reflects comorbidities

Brain inflammation is an important factor in the conversion of a healthy brain into an epileptic one, a phenomenon known as epileptogenesis, offering a new entry point for prognostic tools. The development of anti-epileptogenic therapies to treat before or at disease onset is hampered by our inability to predict the severity of the disease outcome. In a rat model of temporal lobe epilepsy we aimed to assess whether in vivo non-invasive imaging of brain inflammation at disease onset was predictive of spontaneous recurrent seizures (SRS) frequency and severity of depression-like and sensorimotor-related comorbidities. To this end, translocator protein, a biomarker of inflammation, was imaged by means of positron emission tomography (PET) 2 and 4weeks post-status epilepticus using [18F]-PBR111. Translocator protein was highly upregulated 2weeks post-status epilepticus in limbic structures (up to 2.1-fold increase compared to controls in temporal lobe, P<0.001), whereas 4weeks post-status epilepticus, upregulation decreased (up to 1.6-fold increase compared to controls in temporal lobe, P<0.01) and was only apparent in a subset of these regions. Animals were monitored with video-electroencephalography during all stages of disease (acute, latent - first seizures appearing around 2weeks post-status epilepticus - and chronic phases), for a total of 12weeks, in order to determine SRS frequency for each subject (range 0.00-0.83SRS/day). We found that regional PET uptake at 2 and 4weeks post-status epilepticus correlated with the severity of depression-like and sensorimotor-related comorbidities during chronic epilepsy (P<0.05 for each test). Regional PET imaging did not correlate with SRS frequency, however, by applying a multivariate data-driven modeling approach based on translocator protein PET imaging at 2weeks post-status epilepticus, we accurately predicted the frequency of SRS (R=0.92; R2=0.86; P<0.0001) at the onset of epilepsy. This study not only demonstrates non-invasive imaging of translocator protein as a prognostic biomarker to ascertain SRS frequency, but also shows its capability to reflect the severity of depression-like and sensorimotor-related comorbidities. Our results are an encouraging step towards the development of anti-epileptogenic treatments by providing early quantitative assessment of SRS frequency and severity of comorbidities with high clinical relevance.

Performance Characterization of an Actively Cooled Repetitive Transcranial Magnetic Stimulation Coil for the Rat

This study characterizes and validates a recently developed dedicated circular rat coil for small animal repetitive Transcranial Magnetic Stimulation (rTMS).

Rat Brain Normalization Templates for Robust Regional Analysis of [11C]ABP688 Positron Emission Tomography/Computed Tomography

A methodology to generate rat brain templates for spatial normalization of positron emission tomographic (PET)/computed tomographic (CT) images is described and applied to generate three different templates for imaging of [11C]ABP688, a PET ligand binding to the metabotropic glutamate 5 receptor. The templates are based on functional (PET), structural (CT), and combined PET and CT information, respectively. The templates are created from a test–retest study under normal conditions and are used to assess the different templates by using them in the analysis pipeline of a test–retest and a blocking experiment. The resulting average nondisplaceable binding potentials (BPND) show significant (analysis of variance, p < .05) and substantial (up to 23%) differences between the different approaches in several brain regions. The highest BPND values in receptor-rich regions are obtained using the PET-based approach. This approach also had the smallest variability in all tested regions (standard error of measurement of 9% versus 14% [PET/CT] and 20% [CT]). All approaches showed similar relative changes in BPND values with increased blocking. Taken together, these results suggest that the use of the tracer-specific PET-based template outperforms the other approaches with the performance of the combined PET/CT template between those of the PET and the tracer-independent CT template.A methodology to generate rat brain templates for spatial normalization of positron emission tomographic (PET)/computed tomographic (CT) images is described and applied to generate three different templates for imaging of [11C]ABP688, a PET ligand binding to the metabotropic glutamate 5 receptor. The templates are based on functional (PET), structural (CT), and combined PET and CT information, respectively. The templates are created from a test-retest study under normal conditions and are used to assess the different templates by using them in the analysis pipeline of a test-retest and a blocking experiment. The resulting average nondisplaceable binding potentials (BPND) show significant (analysis of variance, p < .05) and substantial (up to 23%) differences between the different approaches in several brain regions. The highest BPND values in receptor-rich regions are obtained using the PET-based approach. This approach also had the smallest variability in all tested regions (standard error of measurement of 9% versus 14% [PET/CT] and 20% [CT]). All approaches showed similar relative changes in BPND values with increased blocking. Taken together, these results suggest that the use of the tracer-specific PET-based template outperforms the other approaches with the performance of the combined PET/CT template between those of the PET and the tracer-independent CT template.

Validation of the semi-quantitative static SUVR method for [18F]-AV45 PET by pharmacokinetic modeling with an arterial input function

Increased brain uptake of 18F-AV45 visualized by PET is a key biomarker for Alzheimer disease (AD). The SUV ratio (SUVR) is widely used for quantification, but is subject to variability based on choice of reference region and changes in cerebral blood flow. Here we validate the SUVR method against the gold standard volume of distribution (VT) to assess cross-sectional differences in plaque load. Methods: Dynamic 60-min 18F-AV45 (291 ± 67 MBq) and 1-min 15O-H2O (370 MBq) scans were obtained in 35 age-matched elderly subjects, including 10 probable AD, 15 amnestic mild cognitive impairment (aMCI), and 10 cognitively healthy controls (HCs). 18F-AV45 VT was determined from 2-tissue-compartment modeling using a metabolite-corrected plasma input function. Static SUVR was calculated at 50–60 min after injection, using either cerebellar gray matter (SUVRCB) or whole subcortical white matter (SUVRWM) as the reference. Additionally, whole cerebellum, pons, centrum semiovale, and a composite region were examined as alternative references. Blood flow was quantified by 15O-H2O SUV. Data are presented as mean ± SEM. Results: There was rapid metabolization of 18F-AV45, with only 35% of unchanged parent remaining at 10 min. Compared with VT, differences in cortical Aβ load between aMCI and AD were overestimated by SUVRWM (+4% ± 2%) and underestimated by SUVRCB (−10% ± 2%). VT correlated better with SUVRWM (Pearson r: from 0.63 for posterior cingulate to 0.89 for precuneus, P < 0.0001) than with SUVRCB (Pearson r: from 0.51 for temporal lobe [P = 0.002] to 0.82 for precuneus [P < 0.0001]) in all tested regions. Correlation results for the alternative references were in between those for CB and WM. 15O-H2O data showed that blood flow was decreased in AD compared with aMCI in cortical regions (−5% ± 1%) and in the reference regions (CB, −9% ± 8%; WM, −8% ± 8%). Conclusion: Increased brain uptake of 18F-AV45 assessed by the simplified static SUVR protocol does not truly reflect Aβ load. However, SUVRWM is better correlated with VT and more closely reflects VT differences between aMCI and AD than SUVRCB.

Preclinical Comparison of the Amyloid-β 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 [11C]Pittsburgh compound B ([11C]PiB) and [18F]florbetaben ([18F]FBB) for preclinical investigations of amyloid-β pathology.