Identification of brain regions predicting epileptogenesis by serial [18F]GE-180 positron emission tomography imaging of neuroinflammation in a rat model of temporal lobe epilepsy.

Excessive activation of inflammatory signaling pathways seems to be a hallmark of epileptogenesis. Positron emission tomography (PET) allows in vivo detection of brain inflammation with spatial information and opportunities for longitudinal follow-up scanning protocols. Here, we assessed whether molecular imaging of the 18 kDa translocator protein (TSPO) can serve as a biomarker for the development of epilepsy. Therefore, brain uptake of [18F]GE-180, a highly selective radioligand of TSPO, was investigated in a longitudinal PET study in a chronic rat model of temporal lobe epilepsy. Analyses revealed that the influence of the epileptogenic insult on [18F]GE-180 brain uptake was most pronounced in the earlier phase of epileptogenesis. Differences were evident in various brain regions during earlier phases of epileptogenesis with [18F]GE-180 standardized uptake value enhanced by 2.1 to 2.7fold. In contrast, brain regions exhibiting differences seemed to be more restricted with less pronounced increases of tracer uptake by 1.8-2.5fold four weeks following status epilepticus and by 1.5-1.8fold in the chronic phase. Based on correlation analysis, we were able to identify regions with a predictive value showing a correlation with seizure development. These regions include the amygdala as well as a cluster of brain areas. This cluster comprises parts of different brain regions, e.g. the hippocampus, parietal cortex, thalamus, and somatosensory cortex. In conclusion, the data provide evidence that [18F]GE-180 PET brain imaging can serve as a biomarker of epileptogenesis. The identification of brain regions with predictive value might facilitate the development of preventive concepts as well as the early assessment of the interventional success. Future studies are necessary to further confirm the predictivity of the approach.

Synergism of perampanel and zonisamide in the rat amygdala kindling model of temporal lobe epilepsy.

OBJECTIVE: Anticonvulsive monotherapy fails to be effective in one third of patients with epilepsy resulting in the need for polytherapy regimens. However, with the still limited knowledge, drug choices for polytherapy remain empirical. Here we report experimental data from a chronic epilepsy model for the combination of perampanel and zonisamide, which can render guidance for clinical studies and individual drug choices. METHODS: The anticonvulsant effects of the combination of perampanel and zonisamide were evaluated in a rat amygdala kindling model. Furthermore, the potential for motor impairment was evaluated. The type of interaction was quantitatively assessed based on isobolographic analysis. RESULTS: When administered alone, zonisamide dose-dependently increased the afterdischarge threshold in fully kindled rats. Moreover, data confirmed efficacy of perampanel to inhibit seizure initiation and progression with an impact on propagation of activity from the focus. Pronounced threshold increases were observed following administration of a constant zonisamide dosage combined with different doses of perampanel. Isobolographic analysis of drug responses, which is based on individual drug dose-effect data, revealed a synergistic interaction substantiating the high efficacy of the combination. Furthermore, rotarod data indicated that the combination has a favorable tolerability profile when zonisamide is coadministered with low doses of perampanel. Plasma concentration analysis argued against a pharmacokinetic interaction as a basis for the synergism. SIGNIFICANCE: The findings clearly indicate a pronounced synergistic anticonvulsant effect for the combination of the noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist perampanel with zonisamide, which modulates voltage-sensitive sodium channels and T-type calcium currents. Consequently, polytherapy using these two antiepileptic drugs might be efficacious for clinical management of partial-onset seizures. The findings indicate that the impact of dose ratios on tolerability needs be taken into account. With regard to conclusions about the extent of the synergism and its implications further antiepileptic drug combinations need to be evaluated allowing direct comparison.

Minocycline fails to exert antiepileptogenic effects in a rat status epilepticus model.

The tetracycline antibiotic minocycline can exert strong anti-inflammatory, antioxidant, and antiapoptotic effects. There is cumulating evidence that epileptogenic brain insults trigger neuroinflammation and anti-inflammatory concepts can modulate the process of epileptogenesis. Based on the mechanisms of action discussed for minocycline, the compound is of interest for intervention studies as it can prevent the polarization of microglia into a pro-inflammatory state. Here, we assessed the efficacy of sub-chronic minocycline administration initiated immediately following an electrically-induced status epilepticus in rats. The treatment did not affect the development of spontaneous seizures. However, minocycline attenuated behavioral long-term consequences of status epilepticus with a reduction in hyperactivity and hyperlocomotion. Furthermore, the compound limited the spatial learning deficits observed in the post-status epilepticus model. The typical status epilepticus-induced neuronal cell loss was evident in the hippocampus and the piriform cortex. Minocycline exposure selectively protected neurons in the piriform cortex and the hilus, but not in the hippocampal pyramidal layer. In conclusion, the data argue against an antiepileptogenic effect of minocycline in adult rats. However, the findings suggest a disease-modifying impact of the tetracycline affecting the development of behavioral co-morbidities, as well as long-term consequences on spatial learning. In addition, minocycline administration resulted in a selective neuroprotective effect. Although strong anti-inflammatory effects have been proposed for minocycline, we could not verify these effects in our experimental model. Considering the multitude of mechanisms claimed to contribute to minocycline's effects, it is of interest to further explore the exact mechanisms underlying the beneficial effects in future studies.

Proteomic profiling of epileptogenesis in a rat model: Focus on inflammation.

Detailed knowledge about the patterns of molecular alterations during epileptogenesis is a presupposition for identifying targets for preventive or disease-modifying approaches, as well as biomarkers of the disease. Large-scale differential proteome analysis can provide unique and novel perspectives based on comprehensive data sets informing about the complex regulation patterns in the disease proteome. Thus, we have completed an elaborate differential proteome analysis based on label-free LC-MS/MS in a rat model of epileptogenesis. Hippocampus and parahippocampal cortex tissues were sampled and analyzed separately at three key time points chosen for monitoring disease development following electrically-induced status epilepticus, namely, the early post-insult phase, the latency phase, and the chronic phase with spontaneous recurrent seizures. We focused the bioinformatics analysis on proteins linked to immune and inflammatory responses, because of the emerging evidence of the specific pathogenic role of inflammatory signalings during epileptogenesis. In the early post-insult and the latency phases, pathway enrichment analysis revealed an extensive over-representation of Toll-like receptor signaling, pro-inflammatory cytokines, heat shock protein regulation, and transforming growth factor beta signaling and leukocyte transendothelial migration. The inflammatory response in the chronic phase proved to be more moderate with differential expression in the parahippocampal cortex exceeding that in the hippocampus. The data sets provide novel information about numerous differentially expressed proteins, which serve as interaction partners or modulators in key disease-associated inflammatory signaling events. Noteworthy, a set of proteins which act as modulators of the ictogenic Toll-like receptor signaling proved to be differentially expressed. In addition, we report novel data demonstrating the regulation of different Toll-like receptor ligands during epileptogenesis. Taken together, the findings deepen our understanding of modulation of inflammatory signaling during epileptogenesis providing an excellent and comprehensive basis for the identification of target and biomarker candidates.

Expression regulation and targeting of the peroxisome proliferator-activated receptor γ following electrically-induced status epilepticus.

The neuroprotective and anti-inflammatory effects of the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone are of particular interest for disease-modifying and antiepileptogenic approaches. We studied the expression of PPARγ and the impact of rosiglitazone on the consequences of status epilepticus (SE) in a rat post-SE model. Immunohistochemical analysis revealed a selective overexpression of PPARγ in the piriform cortex of rats with spontaneous seizures. Rosiglitazone administration initiated following SE failed to exert relevant effects on the development of spontaneous seizures and neuronal cell loss. Whereas spatial learning in the Morris water maze was delayed in SE animals with vehicle administration, the learning curve of rosiglitazone-treated SE rats showed no significant difference to that of controls. The study provides first evidence arguing against a robust antiepileptogenic effect. However, the findings in the spatial learning paradigm indicate disease-modifying effects.

Glutamate-Mediated Down-Regulation of the Multidrug-Resistance Protein BCRP/ABCG2 in Porcine and Human Brain Capillaries.

Breast cancer resistance protein (BCRP) functions as a major molecular gatekeeper at the blood-brain barrier. Considering its impact on access to the brain by therapeutic drugs and harmful xenobiotics, it is of particular interest to elucidate the mechanisms of its regulation. Excessive glutamate concentrations have been reported during epileptic seizures or as a consequence of different brain insults including brain ischemia. Previously, we have demonstrated that glutamate can trigger an induction of the transporter P-glycoprotein. These findings raised the question whether other efflux transporters are affected in a comparable manner. Glutamate exposure proved to down-regulate BCRP transport function and expression in isolated porcine capillaries. The reduction was efficaciously prevented by coincubation with N-methyl-d-aspartate (NMDA) receptor antagonist MK-801. The involvement of the NMDA receptor in the down-regulation of BCRP was further confirmed by experiments showing an effect of NMDA exposure on brain capillary BCRP transport function and expression. Pharmacological targeting of cyclooxygenase-1 and -2 (COX-1 and -2) using the nonselective inhibitor indomethacin, COX-1 inhibitor SC-560, and COX-2 inhibitor celecoxib revealed a contribution of COX-2 activity to the NMDA receptor's downstream signaling events affecting BCRP. Translational studies were performed using human capillaries isolated from surgical specimens of epilepsy patients. The findings confirmed a glutamate-induced down-regulation of BCRP transport activity in human capillaries, which argued against major species differences. In conclusion, our data reveal a novel mechanism of BCRP down-regulation in porcine and human brain capillaries. Moreover, together with previous data sets for P-glycoprotein, the findings point to a contrasting impact of the signaling pathway on the regulation of BCRP and P-glycoprotein. The effect of glutamate and arachidonic acid signaling on BCRP function might have implications for brain drug delivery and for radiotracer brain access in epilepsy patients and patients with other brain insults.

Glutamate-mediated upregulation of the multidrug resistance protein 2 in porcine and human brain capillaries.

As a member of the multidrug-resistance associated protein (MRP) family, MRP2 affects the brain entry of different endogenous and exogenous compounds. Considering the role of this transporter at the blood-brain barrier, the regulation is of particular interest. However, there is limited knowledge regarding the factors that regulate MRP2 in neurologic disease states. Thus, we addressed the hypothesis that MRP2 might be affected by a glutamate-induced signaling pathway that we previously identified as one key mechanism in the regulation of P-glycoprotein. Studies in isolated porcine brain capillaries confirmed that glutamate and N-methyl-d-aspartic acid (NMDA) exposure upregulates expression and function of MPR2. The involvement of the NMDA receptor was further suggested by the fact that the NMDA receptor antagonist MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], as well as the NMDA receptor glycine binding site antagonist L-701,324 [7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolinone], prevented the impact of glutamate. A role of cyclooxygenase-2 was indicated by coincubation with the cyclooxygenase-2 inhibitor celecoxib and the cyclooxygenase-1/-2 inhibitor indomethacin, which both efficaciously abolished a glutamate-induced upregulation of MRP2. Translational studies in human capillaries from surgical specimen demonstrated a relevant MRP2 efflux function and indicated an effect of glutamate exposure as well as its prevention by cyclooxygenase-2 inhibition. Taken together the findings provide first evidence for a role of a glutamate-induced NMDA receptor/cyclooxygenase-2 signaling pathway in the regulation of MRP2 expression and function. The response to excessive glutamate concentrations might contribute to overexpression of MRP2, which has been reported in neurologic diseases including epilepsy. The overexpression might have implications for brain access of various compounds including therapeutic drugs.

Pre-treatment with the NMDA receptor glycine-binding site antagonist L-701,324 improves pharmacosensitivity in a mouse kindling model.

The glycine co-agonist binding site of the N-methyl-D-aspartat (NMDA) receptor is discussed as an interesting target for different central nervous system diseases. Antagonism at this co-agonist site has been suggested as an alternative to the use of non-competitive or competitive NMDA receptor antagonists, which are associated with a pronounced adverse effect profile in chronic epilepsy models and epilepsy patients. In the present study, we addressed the hypothesis that sub-chronic administration of the glycine-binding site antagonist L-701,324 might exert disease-modifying effects in fully kindled mice during a period with frequent seizure elicitation (massive kindling). Moreover, we analyzed whether L-701,324 exposure during this phase affects the subsequent response to an antiepileptic drug. L-701,324 treatment during the massive kindling phase did not affect ictogenesis. Mean seizure severity and cumulative seizure duration proved to be comparable between vehicle- and L-701,324-treated mice. Following withdrawal of L-701,324 seizure thresholds did not differ in a significant manner from those in animals that received vehicle injections. A low dosage of phenobarbital caused a significant increase of the generalized seizure threshold in the L-701,324 pre-treated group, whereas it did not exert a comparable effect in animals that received vehicle during the massive kindling phase. Analysis of P-glycoprotein in the hilus of the hippocampus revealed lower expression rates in L-701,324 pre-treated kindled mice. In conclusion, the data indicate that targeting of the NMDA receptor glycine-binding site does not result in anticonvulsant or disease-modifying effects. However, it might improve antiepileptic drug responses. The findings might be linked to an impact on P-glycoprotein expression. However, future studies are necessary to further evaluate the mechanisms and assess the potential of respective add-on approaches.

Impact of the neural cell adhesion molecule-derived peptide FGL on seizure progression and cellular alterations in the mouse kindling model.

The neural cell adhesion molecule peptide mimetic fibroblast growth loop (FGL) proved to exert neuroprotective, neurotrophic, and anti-inflammatory effects in different in vitro and in vivo experiments. Based on this beneficial efficacy profile, it is currently in clinical development for neurodegenerative diseases and brain insults. Here, we addressed the hypothesis that the peptide might affect development of seizures in a kindling paradigm, as well as associated behavioral and cellular alterations. Both doses tested, 2 and 10 mg/kg FGL, significantly reduced the number of stimulations necessary to induce a generalized seizure. FGL did not exert relevant effects on the behavioral patterns of kindled animals. As expected, kindling increased the hippocampal cell proliferation rate. Whereas the low dose of FGL did not affect this kindling-associated alteration, 10 mg/kg FGL proved to attenuate the expansion of the doublecortin-positive cell population. These data suggest that FGL administration might have an impact on disease-associated alterations in the hippocampal neuronal progenitor cell population. In conclusion, the effects of the peptide mimetic FGL in the kindling model do not confirm a disease-modifying effect with a beneficial impact on the development or course of epilepsy. The results obtained with FGL rather raise some concern regarding a putative effect, which might promote the formation of a hyperexcitable network. Future studies are required to further assess the risks in models with development of spontaneous seizures.

Dynamic regulation of P-glycoprotein in human brain capillaries.

Considering its role as a major blood-brain barrier gatekeeper, the dynamic regulation of the efflux transporter P-glycoprotein is of considerable functional relevance. In particular, disease-associated alterations in transport function might affect central nervous system drug efficacy. Thus, targeting regulatory signaling cascades might render a basis for novel therapeutic approaches. Using capillaries freshly prepared from patient tissue resected during epilepsy surgery, we demonstrate dynamic regulation of P-glycoprotein in human brain capillaries. Glutamate proved to up-regulate P-glycoprotein efflux transport in a significant manner via endothelial NMDA receptors. Both inhibition of cyclooxygenase-2 and antagonism at the glycine-binding site of the NMDA receptor prevented the glutamate-mediated induction of P-glycoprotein transport function in human capillaries. In conclusion, the data argue against species differences in the signaling factors increasing endothelial P-glycoprotein transport function in response to glutamate exposure. Targeting of cyclooxygenase-2 and of the NMDA receptor glycine-binding site was confirmed as an efficacious approach to control P-glycoprotein function. The findings might render a basis for translational development of add-on approaches to improve brain penetration and efficacy of drugs.