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Synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein that binds levetiracetam and is involved in neurotransmission via an unknown mechanism. SV2A-immunoreactivity is reduced in animal models of epilepsy, and in postmortem hippocampi from patients with temporal lobe epilepsy. It is not known if other regions outside the hippocampus are affected in epilepsy, and whether SV2A expression is permanently reduced or regulated over time. In this study, we induced a generalized status epilepticus (SE) by systemic administration of lithium-pilocarpine to adult female rats. The brains from all animals experiencing SE were collected at different time points after the treatment. The radiotracer, [11C]-UCB-J, binds to SV2A with high affinity, and has been used for in vivo imaging as an a-proxy marker for synaptic density. Here we determined the level of tritiated UCB-J binding by semiquantitative autoradiography in the cerebral cortex, hippocampus, thalamus, and hypothalamus, and in cortical subregions. A prominent and highly significant reduction in SV2A binding capacity was observed over the first days after SE in the cerebral cortex and the hippocampus, but not in the thalamus and hypothalamus. The magnitude in reduction was larger and occurred earlier in the hippocampus and the piriform cortex, than in other cortical subregions. Interestingly, in all areas examined, the binding capacity returned to control levels 12 weeks after the SE comparable to the chronic epileptic phase. These data indicate that lithium-pilocarpine-induced epileptogenesis involves both loss and gain of synapses in the in a time-dependent manner.

Background: A wide range of techniques has been developed over the past decades to characterize amyloid-β (Aβ) pathology in mice. Until now, no method has been established to quantify spatial changes in Aβ plaque deposition due to targeted delivery of substances using ALZET® pumps. Objective: Development of a methodology to quantify the local distribution of Aβ plaques after intracerebral infusion of compounds. Methods: We have developed a toolbox to quantify Aβ plaques in relation to intracerebral injection channels using Zeiss AxioVision® and Microsoft Excel® software. For the proof of concept, intracerebral stereotactic surgery was performed in 50-day-old APP-transgenic mice injected with PBS. At the age of 100 days, brains were collected for immunhistological analysis. Results: The toolbox can be used to analyze and evaluate Aβ plaques (number, size, and coverage) in specific brain areas based on their location relative to the point of the injection or the injection channel. The tool provides classification of Aβ plaques in pre-defined distance groups using two different approaches. Conclusion: This new analytic toolbox facilitates the analysis of long-term continuous intracerebral experimental compound infusions using ALZET® pumps. This method generates reliable data for Aβ deposition characterization in relation to the distribution of experimental compounds.

Background: Detailed pathology analysis and morphological quantification is tedious and prone to errors. Automatic image analysis can help to increase objectivity and reduce time. Here, we present the evaluation of the DeePathology STUDIO™ for automatic analysis of histological whole-slide images using machine learning/artificial intelligence. Objective: To evaluate and validate the use of DeePathology STUDIO for the analysis of histological slides at high resolution. Methods: We compared the DeePathology STUDIO and our current standard method using macros in AxioVision for the analysis of amyloid-β (Aβ) plaques and microglia in APP-transgenic mice at different ages. We analyzed density variables and total time invested with each approach. In addition, we correlated Aβ concentration in brain tissue measured by ELISA with the results of Aβ staining analysis. Results: DeePathology STUDIO showed a significant decrease of the time for establishing new analyses and the total analysis time by up to 90%. On the other hand, both approaches showed similar quantitative results in plaque and activated microglia density in the different experimental groups. DeePathology STUDIO showed higher sensitivity and accuracy for small-sized plaques. In addition, DeePathology STUDIO allowed the classification of plaques in diffuse- and dense-packed, which was not possible with our traditional analysis. Conclusion: DeePathology STUDIO substantially reduced the effort needed for a new analysis showing comparable quantitative results to the traditional approach. In addition, it allowed including different objects (categories) or cell types in a single analysis, which is not possible with conventional methods.

Findings of early cerebral amyloid-β deposition in mice after peripheral injection of amyloid-β-containing brain extracts, and in humans following cadaveric human growth hormone treatment raised concerns that amyloid-β aggregates and possibly Alzheimer’s disease may be transmissible between individuals. Yet, proof that Aβ actually reaches the brain from the peripheral injection site is lacking. Here, we use a proteomic approach combining stable isotope labeling of mammals and targeted mass spectrometry. Specifically, we generate 13C-isotope-labeled brain extracts from mice expressing human amyloid-β and track 13C-lysine-labeled amyloid-β after intraperitoneal administration into young amyloid precursor protein-transgenic mice. We detect injected amyloid-β in the liver and lymphoid tissues for up to 100 days. In contrast, injected 13C-lysine-labeled amyloid-β is not detectable in the brain whereas the mice incorporate 13C-lysine from the donor brain extracts into endogenous amyloid-β. Using a highly sensitive and specific proteomic approach, we demonstrate that amyloid-β does not reach the brain from the periphery. Our study argues against potential transmissibility of Alzheimer’s disease while opening new avenues to uncover mechanisms of pathophysiological protein deposition.

Epileptogenesis-associated brain inflammation might be a promising target to prevent or attenuate epileptogenesis. Positron emission tomography (PET) imaging targeting the translocator protein (TSPO) was applied here to quantify effects of different dosing regimens of the anti-inflammatory drug minocycline during the latent phase in two rodent models of epileptogenesis. After induction of epileptogenesis by status epilepticus (SE), rats were treated with minocycline for 7 days (25 or 50 mg/kg) and mice for 5 or 10 days (50 or 100 mg/kg). All animals were subjected to scans at 1 and 2 weeks post-SE. Radiotracer distribution was analyzed and statistical parametric mapping (SPM) was performed, as well as histological analysis of astroglial activation and neuronal cell loss. Atlas-based analysis of [18F]GE180 PET in rats revealed a dose-dependent regional decrease of TSPO expression at 2 weeks post-SE. Results of SPM analysis depicted a treatment effect already at 1 week post-SE in rats treated with the higher minocycline dose. In mice, TSPO PET imaging did not reveal any treatment effects whereas histology identified only a treatment-related reduction in dispersion of dentate gyrus neurons. TSPO PET served as an auspicious tool for temporal monitoring and quantification of anti-inflammatory effects during epileptogenesis. Importantly, the findings underline the need to applying more than one animal model to avoid missing treatment effects. For future studies, the setup is ready to be applied in combination with seizure monitoring to investigate the relationship between individual early treatment response and disease outcome. Electronic supplementary material The online version of this article (10.1007/s13311-020-00834-5) contains supplementary material, which is available to authorized users.

Objective Identification of patients at risk of developing epilepsy before the first spontaneous seizure may promote the development of preventive treatment providing opportunity to stop or slow down the disease. Methods As development of novel radiotracers and on-site setup of existing radiotracers is highly time-consuming and expensive, we used dual-centre in vitro autoradiography as an approach to characterize the potential of innovative radiotracers in the context of epilepsy development. Using brain slices from the same group of rats, we aimed to characterise the evolution of neuroinflammation and expression of inhibitory and excitatory neuroreceptors during epileptogenesis using translational positron emission tomography (PET) tracers; 18 F-flumazenil (18 F-FMZ; GABAA receptor), 18 F-FPEB (metabotropic glutamate receptor 5; mGluR5), 18 F-flutriciclamide (translocator protein; TSPO, microglia activation) and 18 F-deprenyl (monoamine oxidase B, astroglia activation). Autoradiography images from selected time points after pilocarpine-induced status epilepticus (SE; baseline, 24 and 48 hours, 5, 10 and 15 days and 6 and 12-14 weeks after SE) were normalized to a calibration curve, co-registered to an MRI-based 2D region-of-interest atlas, and activity concentration (Bq/mm2 ) was calculated. Results In epileptogenesis-associated brain regions, 18 F-FMZ and 18 F-FPEB showed an early decrease after SE. 18 F-FMZ decrease was maintained in the latent phase and further reduced in the chronic epileptic animals, while 18 F-FPEB signal recovered from day 10, reaching baseline levels in chronic epilepsy. 18 F-flutriciclamide showed an increase of activated microglia at 24 hours after SE, peaking at 5-15 days and decreasing during the chronic phase. On the other hand, 18 F-deprenyl autoradiography showed late astrogliosis, peaking in the chronic phase. Significance Autoradiography revealed different evolution of the selected targets during epileptogenesis. Our results suggest an advantage of combined imaging of inter-related targets like glutamate and GABAA receptors, or microglia and astrocyte activation, in order to identify important interactions, especially when using PET imaging for the evaluation of novel treatments.

Plastic changes in the central auditory system involving the GABAergic system accompany age-related hearing loss. Such processes can be investigated with positron emission tomography (PET) imaging using [18F]flumazenil ([18F]FMZ). Here, [18F]FMZ PET-based modeling approaches allow a simple and reliable quantification of GABAA receptor binding capacity revealing regional differences and age-related changes. Sixty-minute list-mode PET acquisitions were performed in 9 young (range 5–6 months) and 11 old (range 39–42 months) gerbils, starting simultaneously with the injection of [18F]FMZ via femoral vein. Non-displaceable binding potentials (BPnd) with pons as reference region were calculated for auditory cortex (AC), inferior colliculus (IC), medial geniculate body (MGB), somatosensory cortex (SC), and cerebellum (CB) using (i) a two-tissue compartment model (2TCM), (ii) the Logan plot with image-derived blood-input (Logan (BI)), (iii) a simplified reference tissue model (SRTM), and (iv) the Logan reference model (Logan (RT)). Statistical parametric mapping analysis (SPM) comparing young and old gerbils was performed using 3D parametric images for BPnd based on SRTM. Results were verified with in vitro autoradiography from five additional young gerbils. Model assessment included the Akaike information criterion (AIC). Hearing was evaluated using auditory brainstem responses. BPnd differed significantly between models (p

2-Deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has been widely used for imaging brain metabolism. Tracer injection in anesthetized animals is a prerequisite for performing dynamic positron emission tomography (PET) scanning. Since preconditioning, as well as anesthesia, has been described to potentially influence brain [18F] FDG levels, this study evaluated how these variables globally and regionally affect both [18F] FDG uptake and kinetics in murine brain. Sixty-minute dynamic [18F] FDG PET scans were performed in adult male C57BL/6 mice anesthetized with isoflurane [control (in 100 % O2), in medical air, in 100 % O2 + insulin pre-treatment, and in 100 % O2 after 18 h fasting], ketamine/xylazine, sevoflurane, and chloral hydrate. An additional group was scanned after awake uptake. Blood glucose levels were determined, and data was analyzed by comparing percent injected dose per cc tissue (%ID/cc) and glucose influx rate and metabolic rate (MRGlu) calculated by Patlak plot. Ketamine/xylazine and chloral hydrate anesthesia induced a lower whole-brain uptake of [18F] FDG (2.86 ± 0.67 %ID/cc, p

Ischemic stroke imparts elevated risk of heart failure though the underlying mechanisms remain poorly described. We aimed to characterize the influence of cerebral ischemic injury on cardiac function using multimodality molecular imaging to investigate brain and cardiac morphology and tissue inflammation in two mouse models of variable stroke severity. Transient middle cerebral artery occlusion (MCAo) generated extensive stroke damage (56.31 ± 40.39 mm3). Positron emission tomography imaging of inflammation targeting the mitochondrial translocator protein (TSPO) revealed localized neuroinflammation at 7 days after stroke compared to sham (3.8 ± 0.8 vs 2.6 ± 0.7 %ID/g max, p p p = 0.022), but was independent of leukocyte infiltration defined by flow cytometry. Stroke size correlated with severity of cardiac dysfunction (r = 0.590, p = 0.008). Statistical parametric mapping identified a direct association between neuroinflammation at 7 days in a cluster of voxels including the insular cortex and reduced ejection fraction (ρ = − 0.396, p = 0.027). Suppression of microglia led to lower TSPO signal at 7 days which correlated with spared late cardiac function after MCAo (r = − 0.759, p = 0.029). Regional neuroinflammation early after cerebral ischemia influences subsequent cardiac dysfunction. Total body TSPO PET enables monitoring of neuroinflammation, providing insights into brain–heart inter-organ communication and may guide therapeutic intervention to spare cardiac function post-stroke.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG; Ev Studienwerk Villigst Introduction Cerebral ischemia remains a major contributor to global morbidity and mortality. Beyond neurological dysfunction, cerebral stroke also increases risk of subsequent cardiac events and development of chronic heart failure. The mechanisms underlying this brain-heart communication are poorly characterized, but systemic inflammation is thought to play a role. Purpose We hypothesized that cerebral ischemic stroke evokes systemic inflammation contributing to acute cardiac stress and persistent contractile dysfunction, which may be monitored by serial molecular imaging by positron emission tomography (PET). Methods C57Bl6 mice underwent transient (30min) intraluminal middle cerebral artery occlusion (MCAo, n = 88) to induce stroke or sham surgery (n = 18). Serial 18F-GE180 PET at 24h, 7d, and 21d after injury identified translocator protein (TSPO) in brain microglia and peripheral macrophages as a marker of inflammation. Magnetic resonance imaging (MRI) determined stroke size and characterized cardiac function at 1 and 3wk after MCAo. Imaging signals were confirmed by autoradiography, immunohistochemistry and fluorescence-activated cell sorting. Results MCAo in mice induced cerebral stroke of variable size (median 44.68mm3, range:5.12-139.60mm3). Localized inflammation in the injured hemisphere was observed by TSPO PET covering territory consistent to the stroke location, with marked increase in the neuroinflammatory signal at 7d relative to the contralateral hemisphere (% injected dose (ID)/g max: 3.82 ± 0.84 vs 2.51 ± 0.32, p Conclusions Ischemic stroke evoked parallel cerebral and cardiac TSPO upregulation and persistent contractile dysfunction, beginning early after injury. The extent and severity of brain injury predicts left ventricle dysfunction, but the role of inflammation requires further investigation. Molecular imaging may identify risk and guide novel therapies to improve cardiac outcome after stroke.

Background Interest in artificial intelligence-driven analysis of medical images has seen a steep increase in recent years. Thus, our paper aims to promote and facilitate the use of this state-of-the-art technology to fellow researchers and clinicians. New method We present custom deep learning models generated in DeePathology™ STUDIO without the need for background knowledge in deep learning and computer science underlined by practical suggestions. Results We describe the general workflow in this commercially available software and present three real-world examples how to detect microglia on IBA1-stained mouse brain sections including their differences, validation results and analysis of a sample slide. Comparison with existing methods Deep-learning assisted analysis of histological images is faster than classical analysis methods, and offers a wide variety of detection possibilities that are not available using methods based on staining intensity. Conclusions Reduced researcher bias, increased speed and extended possibilities make deep-learning assisted analysis of histological images superior to traditional analysis methods for histological images.

Status epilepticus (SE) is a clinical emergency with high mortality. SE can trigger neuronal death or injury and alteration of neuronal networks resulting in long-term cognitive decline or epilepsy. Among the multiple factors contributing to this damage, imbalance between oxygen and glucose requirements and brain perfusion during SE has been proposed. Herein, we aimed to quantify by neuroimaging the spatiotemporal course of brain perfusion during and after lithium-pilocarpine-induced SE in rats. To this purpose, animals underwent 99mTc-HMPAO SPECT imaging at different time points during and after SE using a small animal SPECT/CT system. 99mTc-HMPAO regional uptake was normalized to the injected dose. In addition, voxel-based statistical parametric mapping was performed. SPECT imaging showed an increase of cortical perfusion before clinical seizure activity onset followed by regional hypo-perfusion starting with the first convulsive seizure and during SE. Twenty-four hours after SE, brain 99mTc-HMPAO uptake was widely decreased. Finally, chronic epileptic animals showed regionally decreased perfusion affecting hippocampus and cortical sub-regions. Despite elevated energy and oxygen requirements, brain hypo-perfusion is present during SE. Our results suggest that insufficient compensation of required blood flow might contribute to the neuronal damage and neuroinflammation, and ultimately to chronic epilepsy generated by SE.

Acute myocardial infarction (MI) triggers a local and systemic inflammatory response. We recently showed microglia involvement using translocator protein imaging. Here, we evaluated whether (11)C-methionine provides further insight into heart–brain inflammation networking. Methods: Male C57BL/6 mice underwent permanent coronary artery ligation followed by (11)C-methionine PET at 3 and 7 d (n = 3). In subgroups, leukocyte homing was blocked by integrin antibodies (n = 5). The cellular substrate for PET signal was identified using brain section immunostaining. Results: (11)C-methionine uptake (percentage injected dose/cm(3)) peaked in the MI region on day 3 (5.9 ± 0.9 vs. 2.4 ± 0.5), decreasing to the control level by day 7 (4.3 ± 0.6). Brain uptake was proportional to cardiac uptake (r = 0.47, P < 0.05), peaking also on day 3 (2.9 ± 0.4 vs. 2.4 ± 0.3) and returning to baseline on day 7 (2.3 ± 0.4). Integrin blockade reduced uptake at every time point. Immunostaining on day 3 revealed colocalization of the l-type amino acid transporter, with glial fibrillary acidic protein–positive astrocytes but not CD68-positive microglia. Conclusion: PET imaging with (11)C-methionine specifically identifies an astrocyte component, enabling further dissection of the heart–brain axis in post-MI inflammation.

Intracerebral administration of the potassium channel blocker 4-aminopyridine (4-AP) triggers neuronal depolarization and intense acute seizure activity followed by neuronal damage. We have recently shown that, in the lithium-pilocarpine rat model of status epilepticus (SE), a single administration of metyrapone, an inhibitor of the 11β-hydroxylase enzyme, had protective properties of preventive nature against signs of brain damage and neuroinflammation. Herein, our aim was to investigate to which extent, pretreatment with metyrapone (150 mg/kg, i.p.) was also able to prevent eventual changes in the acute brain metabolism and short-term neuronal damage induced by intrahippocampal injection of 4-AP (7 μg/5 μl). To this end, regional brain metabolism was assessed by 2-deoxy-2-[18F]fluoro- d -glucose ([18F]FDG) positron emission tomography (PET) during the ictal period. Three days later, markers of neuronal death and hippocampal integrity and apoptosis (Nissl staining, NeuN and active caspase-3 immunohistochemistry), neurodegeneration (Fluoro-Jade C labeling), astrogliosis (glial fibrillary acidic protein (GFAP) immunohistochemistry) and microglia-mediated neuroinflammation (in vitro [18F]GE180 autoradiography) were evaluated. 4-AP administration acutely triggered marked brain hypermetabolism within and around the site of injection as well as short-term signs of brain damage and inflammation. Most important, metyrapone pretreatment was able to reduce ictal hypermetabolism as well as all the markers of brain damage except microglia-mediated neuroinflammation. Overall, our study corroborates the neuroprotective effects of metyrapone against multiple signs of brain damage caused by seizures triggered by 4-AP. Ultimately, our data add up to the consistent protective effect of metyrapone pretreatment reported in other models of neurological disorders of different etiology.

Objective Accumulating evidence suggests that brain inflammation, elicited by epileptogenic insults, is involved in epilepsy development. Noninvasive nuclear imaging of brain inflammation in animal models of epileptogenesis represents a diagnostic in vivo approach with potential for direct translation into the clinic. Here, we investigated up-regulation of the translocator protein (TSPO) indicative of microglial activation by serial [18 F]GE180 positron emission tomographic (PET) imaging in a mouse model of temporal lobe epilepsy. Methods As epileptogenic insult, a status epilepticus (SE) was induced in mice by intrahippocampal injection of kainate. Post-SE mice injected with kainate and sham-injected mice were subjected to [18 F]GE180 PET scans before SE and at 2 days, 5-7 days, 2 weeks, 3 weeks, 7 weeks, and 14 weeks postinsult. For data evaluation, brain regions ipsilateral and contralateral to the injection site were outlined by coregistration with a standard mouse brain atlas, and percentage of injected dose per cubic centimeter was calculated. In addition, a statistical parametric mapping analysis, comparing post-SE mice to baseline, sham mice to baseline, and post-SE to sham mice was performed. Results Following SE, elevations in [18 F]GE180 uptake were most prominent in the ipsilateral hippocampus, occurring between 2 days and at least 7 weeks after SE, with a peak at 5-7 days after SE. In the contralateral hippocampus and other epilepsy-associated brain regions, increased tracer uptake was observed with a similar time profile but to a lesser extent. Moderate enhancement of tracer uptake was also evident in mice after sham surgery. Significance TSPO in vivo imaging reliably detects brain inflammation during epileptogenesis. These inflammatory processes most prominently affect the hippocampus ipsilateral to the injection site. Inflammation induced by the traumatic insult associated with surgery synergistically contributes to total brain inflammation and may also contribute to epileptogenesis. The revealed time course of neuroinflammation will help to identify appropriate time points for anti-inflammatory, potentially antiepileptogenic treatment.

Introduction Alzheimer’s disease (AD) is the leading cause of dementia and a major global health issue. Currently, only limited treatment options are available to patients. One possibility to expand the treatment repertoire is repurposing of existing drugs such as dimethyl fumarate (DMF). DMF is approved for treatment of multiple sclerosis and previous animal studies have suggested that DMF may also have a beneficial effect for the treatment of AD. Methods We used an APPPS1 transgenic model of senile β-amyloidosis and treated female mice orally with DMF in two treatment paradigms (pre and post onset). We quantified learning and memory parameters, β-amyloidosis, and neuroinflammation to determine the potential of DMF as AD therapeutics. Results Treatment with DMF had no influence on water maze performance, β-amyloid accumulation, plaque formation, microglia activation, and recruitment of immune cells to the brain. Compared to vehicle-treated animals, oral DMF treatment could not halt or retard disease progression in the mice. Discussion Our results do not favour the use of DMF as treatment for AD. While our results stand in contrast to previous findings in other models, they emphasize the importance of animal model selection and suggest further studies to elucidate the mechanisms leading to conflicting results.

Background: The recent failure of clinical trials to treat Alzheimer’s disease (AD) indicates that the current approach of modifying disease is either wrong or is too late to be efficient. Mild cognitive impairment (MCI) denotes the phase between the preclinical phase and clinical overt dementia. AD mouse models that overexpress human amyloid-β (Aβ) are used to study disease pathogenesis and to conduct drug development/testing. However, there is no direct correlation between the Aβ deposition, the age of onset, and the severity of cognitive dysfunction. Objective: To detect and predict MCI when Aβ plaques start to appear in the hippocampus of an AD mouse. Methods: We trained wild-type and AD mice in a Morris water maze (WM) task with different inter-trial intervals (ITI) at 3 months of age and assessed their WM performance. Additionally, we used a classification algorithm to predict the genotype (APPtg versus wild-type) of an individual mouse from their respective WM data. Results: MCI can be empirically detected using a short-ITI protocol. We show that the ITI modulates the spatial learning of AD mice without affecting the formation of spatial memory. Finally, a simple classification algorithm such as logistic regression on WM data can give an accurate prediction of the cognitive dysfunction of a specific mouse. Conclusion: MCI can be detected as well as predicted simultaneously with the onset of Aβ deposition in the hippocampus in AD mouse model. The mild cognitive impairment prediction can be used for assessing the efficacy of a treatment.

Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed network-based approaches to prevent epileptogenesis. For proof of concept we combined two drugs (levetiracetam and topiramate) for which in silico analysis of drug-protein interaction networks indicated a synergistic effect on a large functional network of epilepsy-relevant proteins. Using the intrahippocampal kainate mouse model of temporal lobe epilepsy, the drug combination was administered during the latent period before onset of spontaneous recurrent seizures (SRS). When SRS were periodically recorded by video-EEG monitoring after termination of treatment, a significant decrease in incidence and frequency of SRS was determined, indicating antiepileptogenic efficacy. Such efficacy was not observed following single drug treatment. Furthermore, a combination of levetiracetam and phenobarbital, for which in silico analysis of drug-protein interaction networks did not indicate any significant drug-drug interaction, was not effective to modify development of epilepsy. Surprisingly, the promising antiepileptogenic effect of the levetiracetam/topiramate combination was obtained in the absence of any significant neuroprotective or anti-inflammatory effects as indicated by multimodal brain imaging and histopathology. High throughput RNA-sequencing (RNA-seq) of the ipsilateral hippocampus of mice treated with the levetiracetam/topiramate combination showed that several genes that have been linked previously to epileptogenesis, were significantly differentially expressed, providing interesting entry points for future mechanistic studies. Overall, we have discovered a novel combination treatment with promise for prevention of epilepsy.

Rationale Neuronal excitability and brain energy homeostasis are strongly interconnected and evidence suggests that both become altered during epileptogenesis. Pharmacologic modulation of cerebral glucose metabolism might therefore exert anti-epileptogenic effects. Here we provide mechanistic insights into effects of the glycolytic inhibitor 2-deoxy- d -glucose (2-DG) on experimental epileptogenesis by longitudinal 2-deoxy-2[ 18 F]fluoro- d -glucose positron emission tomography ([ 18 F]FDG PET) and histology. Methods To imitate epileptogenesis, 6 Hz-corneal kindling was performed in male NMRI mice by twice daily electrical stimulation for 21 days. Kindling groups were treated i.p. 1 min after each stimulation with either 250 mg/kg 2-DG (CoKi_2-DG) or saline (CoKi_vehicle). A separate group of unstimulated mice was treated with 2-DG (2-DG_only). Dynamic 60-min [ 18 F]FDG PET/CT scans were acquired at baseline and interictally on days 10 and 17 of kindling. [ 18 F]FDG uptake (%injected dose/cc) was quantified in predefined regions of interest (ROI) using a MRI-based brain atlas, and kinetic modelling was performed to evaluate glucose net influx rate K i and glucose metabolic rate MR Glu . Furthermore, statistical parametric mapping (SPM) analysis was applied on kinetic brain maps. For histological evaluation, brain sections were stained for glucose transporter 1 (GLUT1), astrocytes, microglia, as well as dying neurons. Results Post-stimulation 2-DG treatment attenuated early kindling progression, indicated by a reduction of fully-kindled mice, and a lower overall percentage of type five seizures. While 2-DG treatment alone led to globally increased K i and MR Glu values at day 17, kindling progression per se did not influence glucose turnover. Kindling accompanied by 2-DG treatment, however, resulted in regionally elevated [ 18 F]FDG uptake as well as increased K i at days 10 and 17 compared both to baseline and to the 2-DG_only group. In hippocampus and thalamus, higher MR Glu values were found in the CoKi_2-DG vs. the CoKi_vehicle group at day 17. t maps resulting from SPM analysis generally confirmed the results of the ROI analysis, and additionally revealed increased MR Glu restricted to the ventral hippocampus when comparing the CoKi_2-DG and the 2-DG_only group both at days 10 and, more distinct, day 17. Immunohistochemical staining showed an attenuated kindling-induced regional activation of astrocytes in the CoKi_2-DG group. Interestingly, 2-DG treatment alone (and also in combination with kindling, but not kindling alone) led to increased microglial activation scores, whereas neither staining of GLUT1 nor of dying neurons revealed any differences to untreated controls. Conclusions Post-stimulation treatment with 2-DG exerts disease-modifying effects in the mouse 6 Hz corneal kindling model. The observed local increase in glucose supply and turnover, the alleviation of astroglial activation and the activation of microglia by 2-DG might contribute separately or in combination to its positive interference with epileptogenesis.

Objective Acquired epilepsy is a devastating long-term risk of various brain insults, including trauma, stroke, infections, and status epilepticus (SE). There is no preventive treatment for patients at risk. Attributable to the complex alterations involved in epileptogenesis, it is likely that multitargeted approaches are required for epilepsy prevention. We report novel preclinical findings with isoflurane, which exerts various nonanesthetic effects that may be relevant for antiepileptogenesis. Methods The effects of isoflurane were investigated in two rat models of SE-induced epilepsy: intrahippocampal kainate and systemic administration of paraoxon. Isoflurane was either administered during (kainate) or after (paraoxon) induction of SE. Magnetic resonance imaging was used to assess blood–brain barrier (BBB) dysfunction. Positron emission tomography was used to visualize neuroinflammation. Long-term electrocorticographic recordings were used to monitor spontaneous recurrent seizures. Neuronal damage was assessed histologically. Results In the absence of isoflurane, spontaneous recurrent seizures were common in the majority of rats in both models. When isoflurane was administered during kainate injection, duration and severity of SE were not affected, but only few rats developed spontaneous recurrent seizures. A similar antiepileptogenic effect was found when paraoxon-treated rats were exposed to isoflurane after SE. Moreover, in the latter model, isoflurane prevented BBB dysfunction and neurodegeneration, whereas isoflurane reduced neuroinflammation in the kainate model. Interpretation Given that isoflurane is a widely used volatile anesthetic, and is used for inhalational long-term sedation in critically ill patients at risk to develop epilepsy, our findings hold a promising potential to be successfully translated into the clinic. Ann Neurol 2016;80:896–908

Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.

Glucose metabolism and serotonergic neurotransmission have been reported to play an important role in epileptogenesis. We therefore aimed to use neuroimaging to evaluate potential alterations in serotonin 5-HT1A receptor and glucose metabolism during epileptogenesis in the rat electrical kindling model. To achieve this goal, we performed positron emission tomography (PET) imaging in a rat epileptogenesis model triggered by electrical stimulation of the hippocampus using 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG), a radiolabeled analog of glucose, and 2'-methoxyphenyl-(N-2'-pyridinyl)-p-18F-fluoro-benzamidoethylpiperazine (18F-MPPF), a radiolabeled 5-HT1A receptor ligand, to evaluate brain metabolism and 5-HT1A receptor functionality. Since the 5-HT1A receptor is also highly expressed in astrocytes, glial fibrillary acidic protein (GFAP) immunofluorescence was performed to detect astrogliosis arising from the kindling procedure once the study was finalized. Lastly, in vitro18F-MPPF autoradiography was performed to evaluate changes in 5HT1A receptor expression. 18F-FDG PET showed reduction of glucose uptake in cortical structures, whereas 18F-MPPF PET revealed an enhancement of tracer binding potential (BPND) in key areas rich in 5-HT1A receptor involved in epilepsy, including septum, hippocampus and entorhinal cortex of kindled animals compared to controls. However, in vitro 5-HT1A receptor autoradiography showed no changes in densitometric signal in any brain region, suggesting that the augmentation in BPND found by PET could be caused by reduction of synaptic serotonin. Importantly, astroglial activation was detected in the hippocampus of kindled rats. Overall, electrical kindling induced hypometabolism, astrogliosis and serotonergic alterations in epilepsy-related regions. Furthermore, the present findings point to 5-HT1A receptor as a valuable epileptogenesis biomarker candidate and a potential therapeutic target.

Alterations in metabolism during epileptogenesis may be a therapy target. Recently, an increase in amino acid transport into the brain was proposed to play a role in epileptogenesis. We aimed to characterize alterations of substrate utilization during epileptogenesis and in chronic epilepsy. The lithium-pilocarpine post status epilepticus (SE) rat model was used. We performed longitudinal O-(2-[(18)F]fluoroethyl)-l-tyrosine (18F-FET) and18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and calculated18F-FET volume of distribution (Vt) and18F-FDG uptake. Correlation analyses were performed with translocator protein-PET defined neuroinflammation from previously acquired data. We found reduced18F-FET Vtat 48 h after SE (amygdala: −30.2%, p = 0.014), whereas18F-FDG showed increased glucose uptake 4 and 24 h after SE (hippocampus: + 43.6% and +42.5%, respectively; p 18F-FET and18F-FDG in the hippocampus. No correlation was found for18F-FET or18F-FDG to microglial activation at seven days post SE. Whereas metabolic alterations do not reflect higher metabolism associated to activated microglia, they might be partially driven by chronic neuronal loss. However, both metabolisms diverge during early epileptogenesis, pointing to amino acid turnover as a possible biomarker and/or therapeutic target for epileptogenesis.

Background and Purpose— This pilot study aims to demonstrate the feasibility of targeting molecular characteristics of high-risk atherosclerotic plaque in symptomatic and asymptomatic carotid stenosis (CS), that is, upregulation of the translocator protein (TSPO) and the chemokine receptor type 4 (CXCR4), by means of molecular imaging. Methods— In a translational setting, specimens of carotid plaques of patients with symptomatic and asymptomatic CS obtained by carotid endarterectomy were analyzed for the presence of TSPO and CXCR4 by autoradiography, using the positron emission tomography tracers 18 F-GE180 and 68 Ga-Pentixafor and evaluated by histopathology. In addition, 68 Ga-Pentixafor positron emission tomography/computed tomography was performed in a patient with high-grade CS. Results— Distinct patterns of upregulation of TSPO ( 18 F-GE180 uptake) and CXCR4 ( 68 Ga-Pentixafor uptake) were identified in carotid plaque by autoradiography. The spatial distribution was associated with specific histological hallmarks that are established features of high-risk plaque: TSPO upregulation correlated with activated macrophages infiltration, whereas CXCR4 upregulation also corresponded to areas of intraplaque hemorrhage. 68 Ga-Pentixafor uptake was significantly higher in plaques of symptomatic compared with asymptomatic CS. Clinical positron emission tomography revealed marked 68 Ga-Pentixafor uptake in carotid plaque of a patient with high-grade CS. Conclusions— Clinical imaging of molecular signatures of high-risk atherosclerotic plaque is feasible and may become a promising diagnostic tool for comprehensive characterization of carotid disease. This methodology provides a platform for future studies targeting carotid plaque.

p-Chloroamphetamine (PCA) is a neurotoxin that selectively degenerates the serotonin (5-HT) axon terminals. In order to study the brain metabolic consequences induced by serotonergic denervation, a single dose of PCA (2.5 or 10 mg/kg i.p.) was administered to male adult rats. In vivo regional brain metabolism was evaluated 3 and 21 days after PCA (2.5 or 10 mg/kg; i.p.) injection by 2-deoxy-2-[18F] fluoro-d-glucose ([18F] FDG) positron emission tomography (PET). At day 22, the following markers of neurotoxicity were determined: (a) 5-HT axon terminal lesion by 5-HT transporter (SERT) autoradiography, (b) reactive gliosis by glial fibrillary acidic protein immunohistochemistry, and (c) eventual neurodegeneration by DAPI/Fluoro-Jade C labeling. An average of 20 % reduction of [18F] FDG uptake in most brain areas was observed at day 21 under 10 mg/kg PCA treatment. Instead, 2.5 mg/kg PCA only reduced metabolic activity in neocortex. Likewise, the high dose of PCA exerted a strong decrease (>30 %) in SERT density in several 5-HT innervated regions, but no effect was found in midbrain raphe nuclei, the main source of serotonergic neurons. Although PCA induced astroglial activation both in hippocampus and cortex in response to axotomy, no signs of neuronal death in these areas were detected. Overall, [18F] FDG PET revealed that the reduction of the brain metabolic activity induced by PCA is related to 5-HT axon terminal lesion, with no apparent affectation of neuronal viability.

The status epilepticus (SE) induced by lithium-pilocarpine is a well characterized rodent model of the human temporal lobe epilepsy (TLE) which is accompanied by severe brain damage. Stress and glucocorticoids markedly contribute to exacerbate neuronal damage induced by seizures but the underlying mechanisms are poorly understood. Herein we sought to investigate whether a single administration of metyrapone (150 mg/kg, i.p.), an 11β-hydroxylase inhibitor, enzyme involved in the peripheral and central synthesis of corticosteroids, had neuroprotective properties in this model. Two experiments were carried out. In exp. 1, metyrapone was administered 3 h before pilocarpine injection whereas in exp. 2, metyrapone administration took place at the onset of the SE. In both experiments, 3 days after the insult, brain metabolism was assessed by in vivo 2-deoxy-2-[18F]fluoro- d -glucose ([18F]FDG) positron emission tomography (PET). Brains were processed for analyses of markers of hippocampal integrity (Nissl staining), neurodegeneration (Fluoro-Jade C), astrogliosis (glial fibrillary acidic protein (GFAP) immunohistochemistry) and, for a marker of activated microglia by in vitro autoradiography with the TSPO (18 kDa translocator protein) radioligand [18F]GE180. The SE resulted in a consistent hypometabolism in hippocampus, cortex and striatum and neuronal damage, hippocampal neurodegeneration, neuronal death and gliosis. Interestingly, metyrapone had neuroprotective effects when administered before, but not after the insult. In summary, we conclude that metyrapone administration prior but not after the SE protected from brain damage induced by SE in the lithium-pilocarpine model. Therefore, it seems that the effect of metyrapone is preventive in nature and likely related to its antiseizure properties.

Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.

Epileptogenesis, i.e., development of epilepsy, involves a number of processes that alter the brain function in the way that triggers spontaneous seizures. Kindling is one of the most used animal models of temporal lobe epilepsy (TLE) and epileptogenesis, although chemical kindling suffers from high inter-assay success unpredictability. This study was aimed to analyze the eventual regional brain metabolic changes during epileptogenesis in the pentylenetetrazole (PTZ) kindling model in order to obtain a predictive kindling outcome parameter. In vivo longitudinal positron emission tomography (PET) scans with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) along the PTZ kindling protocol (35 mg/kg intraperitoneally (i.p.), 18 sessions) in adult male rats were performed in order to evaluate the regional brain metabolism. The half of the PTZ-injected rats reached the kindled state. In addition, a significant decrease of [18F]FDG uptake at the end of the protocol in most of the brain structures of kindled animals was found, reflecting the characteristic epilepsy-associated hypometabolism. However, PTZ-injected animals but not reaching the kindled state did not show this widespread brain hypometabolism. Retrospective analysis of the data revealed that hippocampal [18F]FDG uptake normalized to pons turned out to be a predictive index of the kindling outcome. Thus, a 19.06 % reduction (p = 0.008) of the above parameter was found in positively kindled rats compared to non-kindled ones just after the fifth PTZ session. Non-invasive PET neuroimaging was a useful tool for discerning epileptogenesis progression in this animal model. Particularly, the [18F]FDG uptake of the hippocampus proved to be an early predictive parameter to differentiate resistant and non-resistant animals to the PTZ kindling.

Experimental and clinical evidence suggests that neuroinflammation, triggered by epileptogenic insults, contributes to seizure development. We used translocator protein–targeted molecular imaging to obtain further insights into the role of microglial activation during epileptogenesis. Methods: As epileptogenic insult, a status epilepticus (SE) was induced in rats by lithium pilocarpine. Rats were subjected to 11C-PK11195 PET scans before SE; at 4 h after SE; at 1, 2, 5, 7, 14, and 22 d after SE; and at 14–16 wk after SE. For data evaluation, brain regions were outlined by coregistration with a standard rat brain atlas, and percentage injected dose/cm3 and binding potential (simplified reference tissue model with cerebellar gray matter as a reference region) were calculated. For autoradiography and immunohistochemical evaluation, additional rats were decapitated without prior SE or 2, 5, or 14 d after SE. Results: After SE, increases in 11C-PK11195 uptake and binding potential were evident in epileptogenesis-associated brain regions, such as the hippocampus, thalamus, or piriform cortex, but not in the cerebellum beginning at 2–5 d and persisting at least 3 wk after SE. Maximal regional signal was observed at 1–2 wk after SE. Autoradiography confirmed the spatiotemporal profile. Immunohistochemical evaluation revealed microglial and astroglial activation as well as neuronal cell loss in epileptogenesis-associated brain regions at all investigated time points. The time course of microglial activation was consistent with that demonstrated by tracer techniques. Conclusion: Translocator protein–targeted PET is a reliable tool for identifying brain inflammation during epileptogenesis. Neuroinflammation mainly affects brain regions commonly associated with seizure generation and spread. Definition of the time profile of neuroinflammation may facilitate the development of inflammation-targeted, antiepileptogenic therapy.

Reducing insulin-like growth factor I receptor (IGF-IR) levels or administration of IGF-I show beneficial effects in the brain. We now provide evidence to help resolve this paradox. The unliganded IGF-IR inhibits glucose uptake by astrocytes while its stimulation with IGF-I, in concert with insulin activation of the insulin receptor, produces the opposite effect. In vivo imaging showed that shRNA interference of brain IGF-IR increased glucose uptake by astrocytes while pharmacological blockade of IGF-IR reduced it. Brain 18FGlucose-PET of IGF-IR shRNA injected mice confirmed an inhibitory role of unliganded IGF-IR on glucose uptake, whereas glucose-dependent recovery of neuronal activity in brain slices was blunted by pharmacological blockade of IGF-IR. Mechanistically, we found that the unliganded IGF-IR retains glucose transporter 1 (GLUT1), the main glucose transporter in astrocytes, inside the cell while IGF-I, in cooperation with insulin, synergistically stimulates MAPK/PKD to promote association of IGF-IR with GLUT 1 via Rac1/GIPC1 and increases GLUT1 availability at the cell membrane. These findings identify IGF-I and its receptor as antagonistic modulators of brain glucose uptake.

It has been reported that fluoxetine, a selective serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitor, has neuroprotective properties in the lithium–pilocarpine model of status epilepticus (SE) in rats. The aim of the present study was to investigate the effect of 5-HT depletion by short-term administration of p-chlorophenylalanine (PCPA), a specific tryptophan hydroxylase inhibitor, on the brain hypometabolism and neurodegeneration induced in the acute phase of this SE model. Our results show that 5-HT depletion did modify neither the brain basal metabolic activity nor the lithium–pilocarpine-induced hypometabolism when evaluated 3 days after the insult. In addition, hippocampal neurodegeneration and astrogliosis triggered by lithium–pilocarpine were not exacerbated by PCPA treatment. These findings point out that in the early latent phase of epileptogenesis, non-5-HT-mediated actions may contribute, at least in some extent, to the neuroprotective effects of fluoxetine in this model of SE.

The role of serotonin (5-hydroxytryptamine; 5-HT) in epileptogenesis still remains controversial. In this regard, it has been reported that serotonergic drugs can alter epileptogenesis in opposite ways. The main objective of this work was to investigate the effect of the selective 5-HT selective reuptake inhibitor (SSRI) fluoxetine administered subacutely (10 mg/kg/day × 7 days) on the eventual metabolic impairment induced by the lithium-pilocarpine model of epilepsy in rats. In vivo 2-deoxy-2-[ 18 F]fluoro- d -glucose ([ 18 F] FDG) positron emission tomography (PET) was performed to assess the brain glucose metabolic activity on days 3 and 30 after the insult. In addition, at the end of the experiment (day 33), several histochemical and neurochemical assessments were performed for checking the neuronal functioning and integrity. Three days after the insult, a marked reduction of [ 18 F] FDG uptake (about 30% according to the brain region) was found in all brain areas studied. When evaluated on day 30, although a hypometabolism tendency was observed, no statistically significant reduction was present in any region analyzed. In addition, lithium-pilocarpine administration was associated with medium-term hippocampal and cortical damage, since it induced neurodegeneration, glial activation and augmented caspase-9 expression. Regarding the effect of fluoxetine, subacute treatment with this SSRI did not significantly reduce the mortality rate observed after pilocarpine-induced seizures. However, fluoxetine did prevent not only the short-term metabolic impairment, but also the aforementioned signs of neuronal damage in surviving animals to lithium-pilocarpine protocol. Finally, fluoxetine increased the density of GABA A receptor both at the level of the dentate gyrus and CA1–CA2 regions in pilocarpine-treated animals. Overall, our data suggest a protective role for fluoxetine against pilocarpine-induced brain damage. Moreover, this action may be associated with an increase of GABA A receptor expression in hippocampus.

p-Chloroamphetamine (PCA) is a neurotoxin that selectively degenerates the serotonin (5-HT) axon terminals. In order to study the brain metabolic consequences induced by serotonergic denervation, a single dose of PCA (2.5 or 10 mg/kg i.p.) was administered to male adult rats.In vivo regional brain metabolism was evaluated 3 and 21 days after PCA (2.5 or 10 mg/kg; i.p.) injection by 2-deoxy-2-[(18)F] fluoro-D-glucose ([(18)F] FDG) positron emission tomography (PET). At day 22, the following markers of neurotoxicity were determined: (a) 5-HT axon terminal lesion by 5-HT transporter (SERT) autoradiography, (b) reactive gliosis by glial fibrillary acidic protein immunohistochemistry, and (c) eventual neurodegeneration by DAPI/Fluoro-Jade C labeling.An average of 20 % reduction of [(18)F] FDG uptake in most brain areas was observed at day 21 under 10 mg/kg PCA treatment. Instead, 2.5 mg/kg PCA only reduced metabolic activity in neocortex. Likewise, the high dose of PCA exerted a strong decrease (30 %) in SERT density in several 5-HT innervated regions, but no effect was found in midbrain raphe nuclei, the main source of serotonergic neurons. Although PCA induced astroglial activation both in hippocampus and cortex in response to axotomy, no signs of neuronal death in these areas were detected.Overall, [(18)F] FDG PET revealed that the reduction of the brain metabolic activity induced by PCA is related to 5-HT axon terminal lesion, with no apparent affectation of neuronal viability.