Selective inhibition of mTORC1/2 or PI3K/mTORC1/2 signaling does not prevent or modify epilepsy in the intrahippocampal kainate mouse model.

Dysregulation of the PI3K/Akt/mTOR pathway has been implicated in several brain disorders, including epilepsy. Rapamycin and similar compounds inhibit mTOR. complex 1 and have been reported to decrease seizures, delay seizure development, or prevent epileptogenesis in different animal models of genetic or acquired epilepsies. However, data for acquired epilepsy are inconsistent, which, at least in part, may be due to the poor brain penetration and long brain persistence of rapamycin and the fact that it blocks only one of the two cellular mTOR complexes. Here we examined the antiepileptogenic or disease-modifying effects of two novel, brain-permeable and well tolerated 1,3,5-triazine derivatives, the ATP-competitive mTORC1/2 inhibitor PQR620 and the dual pan-PI3K/mTORC1/2 inhibitor PQR530 in the intrahippocampal kainate mouse model, in which spontaneous seizures develop after status epilepticus (SE). Following kainate injection, the two compounds were administered over 2 weeks at doses previously been shown to block mTORC1/2 or PI3K/mTORC1/2 in the mouse brain. When spontaneous seizures were recorded by continuous (24/7) video-EEG recording starting 6 weeks after termination of treatment, no effects on incidence or frequency of seizures were observed. Drug treatment suppressed the epilepsy-induced activation of the PI3K/Akt/mTOR pathway in the hippocampus, but granule cell dispersion in the dentate gyrus was not prevented. When epilepsy-associated behavioral alterations were determined 12-14 weeks after kainate, mice pretreated with PQR620 or PQR530 exhibited reduced anxiety-related behavior in the light-dark box, indicating a disease-modifying effect. Overall, the data indicate that mTORC1/C2 or PI3K/mTORC1/C2 inhibition may not be an antiepileptogenic strategy for SE-induced epilepsy.

A face-to-face comparison of the intra-amygdala and intrahippocampal kainate mouse models of mesial temporal lobe epilepsy and their utility for testing novel therapies.

OBJECTIVE: Intracranial (intrahippocampal or intra-amygdala) administration of kainate in rodents leads to spatially restricted brain injury and development of focal epilepsy with characteristics that resemble mesial temporal lobe epilepsy. Such rodent models are used both in the search for more effective antiseizure drugs (ASDs) and in the development of antiepileptogenic strategies. However, it is not clear which of the models is best suited for testing different types of epilepsy therapies. METHODS: In the present study, we performed a face-to-face comparison of the intra-amygdala kainate (IAK) and intrahippocampal kainate (IHK) mouse models using the same mouse inbred strain (C57BL/6). For comparison, some experiments were performed in mouse outbred strains. RESULTS: Intra-amygdala kainate injection led to more severe status epilepticus and higher mortality than intrahippocampal injection. In male C57BL/6 mice, the latent period to spontaneous recurrent seizures (SRSs) was short or absent in both models, whereas a significantly longer latent period was determined in NMRI and CD-1 outbred mice. When SRSs were recorded from the ipsilateral hippocampus, relatively frequent electroclinical seizures were determined in the IAK model, whereas only infrequent electroclinical seizures but extremely frequent focal electrographic seizures were determined in the IHK model. As a consequence of the differences in SRS frequency, prolonged video-electroencephalographic monitoring and drug administration were needed for testing efficacy of the benchmark ASD carbamazepine in the IAK model, whereas acute drug testing was possible in the IHK model. In both models, carbamazepine was only effective at high doses, indicating ASD resistance to this benchmark drug. SIGNIFICANCE: We found a variety of significant differences between the IAK and IHK models, which are important when deciding which of these models is best suited for studies on novel epilepsy therapies. The IAK model appears particularly interesting for studies on disease-modifying treatments, whereas the IHK model is well suited for studying the antiseizure activity of novel ASDs against difficult-to-treated focal seizures.

Macrophage depletion by liposome-encapsulated clodronate suppresses seizures but not hippocampal damage after acute viral encephalitis.

Viral encephalitis is a major risk factor for the development of seizures and epilepsy, but the underlying mechanisms are only poorly understood. Mouse models such as viral encephalitis induced by intracerebral infection with Theiler's virus in C57BL/6 (B6) mice allow advancing our understanding of the immunological and virological aspects of infection-induced seizures and their treatment. Previous studies using the Theiler's virus model in B6 mice have indicated that brain-infiltrating inflammatory macrophages and the cytokines released by these cells are key to the development of acute seizures and hippocampal damage in this model. However, approaches used to prevent or reduce macrophage infiltration were not specific, so contribution of other mechanisms could not be excluded. In the present study, we used a more selective and widely used approach for macrophage depletion, i.e., systemic administration of clodronate liposomes, to study the contribution of macrophage infiltration to development of seizures and hippocampal damage. By this approach, almost complete depletion of monocytic cells was achieved in spleen and blood of Theiler's virus infected B6 mice, which was associated with a 70% decrease in the number of brain infiltrating macrophages as assessed by flow cytometry. Significantly less clodronate liposome-treated mice exhibited seizures than liposome controls (P<0.01), but the development of hippocampal damage was not prevented or reduced. Clodronate liposome treatment did not reduce the increased Iba1 and Mac3 labeling in the hippocampus of infected mice, indicating that activated microglia may contribute to hippocampal damage. The unexpected mismatch between occurrence of seizures and hippocampal damage is thought-provoking and suggests that the mechanisms involved in degeneration of specific populations of hippocampal neurons in encephalitis-induced epilepsy are more complex than previously thought.

Viral mouse models of multiple sclerosis and epilepsy: Marked differences in neuropathogenesis following infection with two naturally occurring variants of Theiler's virus BeAn strain.

Following intracerebral inoculation, the BeAn 8386 strain of Theiler's virus causes persistent infection and inflammatory demyelinating encephalomyelitis in the spinal cord of T-cell defective SJL/J mice, which is widely used as a model of multiple sclerosis. In contrast, C57BL/6 (B6) mice clear the virus and develop inflammation and lesions in the hippocampus, associated with acute and chronic seizures, representing a novel model of viral encephalitis-induced epilepsy. Here we characterize the geno- and phenotype of two naturally occurring variants of BeAn (BeAn-1 and BeAn-2) that can be used to further understand the viral and host factors involved in the neuropathogenesis in B6 and SJL/J mice. Next generation sequencing disclosed 15 single nucleotide differences between BeAn-1 and BeAn-2, of which 4 are coding changes and 3 are in the 5'-UTR (5'-untranslated region). The relatively minor variations in the nucleotide sequence of the two BeAn substrains led to marked differences in neurovirulence. In SJL/J mice, inflammatory demyelination in the spinal cord and its clinical consequences were significantly more marked following infection with BeAn-1 than with BeAn-2. Both BeAn substrains caused lymphocyte infiltration and increase of MAC3-positive cells in the hippocampus, but hippocampal damage and seizures were only observed in B6 mice. Seizures occurred in one third of BeAn-2 infected B6 mice, but not in BeAn-1 infected B6 mice. By comparing individual mice by receiver operating characteristic (ROC) curve analysis, the severity of hippocampal neurodegeneration and amount of MAC3-positive microglia/macrophages discriminated seizing from non-seizing B6 mice, whereas T-lymphocyte brain infiltration was not found to be a crucial factor. These data add novel evidence to the view that differential outcome of infection may be not invariably linked to a distinct viral burden but to a finely tuned balance between antiviral immune responses that although essential for host resistance can also contribute to immunopathology.

Brain inflammation, neurodegeneration and seizure development following picornavirus infection markedly differ among virus and mouse strains and substrains.

Infections, particularly those caused by viruses, are among the main causes of acquired epilepsy, but the mechanisms causing epileptogenesis are only poorly understood. As a consequence, no treatment exists for preventing epilepsy in patients at risk. Animal models are useful to study epileptogenesis after virus-induced encephalitis and how to interfere with this process, but most viruses that cause encephalitis in rodents are associated with high mortality, so that the processes leading to epilepsy cannot be investigated. Recently, intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 (B6) mice was reported to induce early seizures and epilepsy and it was proposed that the TMEV mouse model represents the first virus infection-driven animal model of epilepsy. In the present study, we characterized this model in two B6 substrains and seizure-resistant SJL/J mice by using three TMEV (sub)strains (BeAn-1, BeAn-2, DA). The idea behind this approach was to study what is and what is not necessary for development of acute and late seizures after brain infection in mice. Receiver operating characteristic (ROC) curve analysis was used to determine which virus-induced brain alterations are associated with seizure development. In B6 mice infected with different TMEV virus (sub)strains, the severity of hippocampal neurodegeneration, amount of MAC3-positive microglia/macrophages, and expression of the interferon-inducible antiviral effector ISG15 were almost perfect at discriminating seizing from non-seizing B6 mice, whereas T-lymphocyte brain infiltration was not found to be a crucial factor. However, intense microglia/macrophage activation and some hippocampal damage were also observed in SJL/J mice. Overall, the TMEV model provides a unique platform to study virus and host factors in ictogenesis and epileptogenesis.