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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.

Fingolimod is an approved treatment for relapsing–remitting multiple sclerosis (MS), and its properties in different pathways have raised interest in therapy research for other neurodegenerative diseases. Fingolimod is an agonist of sphingosine-1-phosphate (S1P) receptors. Its main pharmacologic effect is immunomodulation by lymphocyte homing, thereby reducing the numbers of T and B cells in circulation. Because of the ubiquitous expression of S1P receptors, other effects have also been described. Here, we review preclinical experiments evaluating the effects of treatment with fingolimod in neurodegenerative diseases other than MS, such as Alzheimer’s disease or epilepsy. Fingolimod has shown neuroprotective effects in different animal models of neurodegenerative diseases, summarized here, correlating with increased brain-derived neurotrophic factor and improved disease phenotype (cognition and/or motor abilities). As expected, treatment also induced reductions in different neuroinflammatory markers because of not only inhibition of lymphocytes but also direct effects on astrocytes and microglia. Furthermore, fingolimod treatment exhibited additional effects for specific neurodegenerative disorders, such as reduction of amyloid-β production, and antiepileptogenic properties. The neuroprotective effects exerted by fingolimod in these preclinical studies are reviewed and support the translation of fingolimod into clinical trials as treatment in neurodegenerative diseases beyond neuroinflammatory conditions (MS).

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.

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.

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.

Purpose Alterations in brain glucose metabolism detected by 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) positron emission tomography (PET) may serve as an early predictive biomarker and treatment target for epileptogenesis. Here, we aimed to investigate changes in cerebral glucose metabolism before induction of epileptogenesis, during epileptogenesis as well as during chronic epilepsy. As anesthesia is usually unavoidable for preclinical PET imaging and influences the distribution of the radiotracer, four different protocols were compared. Procedures We investigated 18F-FDG uptake phase in conscious rats followed by a static scan as well as dynamic scans under continuous isoflurane, medetomidine-midazolam-fentanyl (MMF), or propofol anesthesia. Furthermore, we applied different analysis approaches: atlas-based regional analysis, statistical parametric mapping, and kinetic analysis. Results At baseline and compared to uptake in conscious rats, isoflurane and propofol anesthesia resulted in decreased cortical 18F-FDG uptake while MMF anesthesia led to a globally decreased tracer uptake. During epileptogenesis, MMF anesthesia was clearly best distinctive for visualization of prominently increased glucometabolism in epilepsy-related brain areas. Kinetic modeling further increased sensitivity, particularly for continuous isoflurane anesthesia. During chronic epilepsy, hypometabolism affecting more or less the whole brain was detectable with all protocols. Conclusion This study reveals evaluation of anesthesia protocols for preclinical 18F-FDG PET imaging as a critical step in the study design. Together with an appropriate data analysis workflow, the chosen anesthesia protocol may uncover otherwise concealed disease-associated regional glucometabolic changes.

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

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.

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.

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.