Development of an MR-Guided Focused Ultrasound (MRgFUS) Lesioning Approach for the Fornix in the Rat Brain.

OBJECTIVE: High-intensity magnetic resonance-guided focused ultrasound (MRgFUS) is a non-invasive therapy to lesion brain tissue, used clinically in patients and pre-clinically in several animal models. Challenges with focused ablation in rodent brains can include skull and near-field heating and accurately targeting small and deep brain structures. We overcame these challenges by creating a novel method consisting of a craniectomy skull preparation, a high-frequency transducer (3 MHz) with a small ultrasound focal spot, a transducer positioning system with an added manual adjustment of ∼0.1 mm targeting accuracy, and MR acoustic radiation force imaging for confirmation of focal spot placement. METHODS: The study consisted of two main parts. First, two skull preparation approaches were compared. A skull thinning approach (n = 7 lesions) was compared to a craniectomy approach (n = 22 lesions), which confirmed a craniectomy was necessary to decrease skull and near-field heating. Second, the two transducer positioning systems were compared with the fornix chosen as a subcortical ablation target. We evaluated the accuracy of targeting using histologic methods from a high-frequency transducer with a small ultrasound focal spot and MR acoustic radiation force imaging. RESULTS: Comparing a motorized adjustment system (∼1 mm precision, n = 17 lesions) to the motorized system with an added micromanipulator (∼0.1 mm precision, n = 14 lesions), we saw an increase in the accuracy of targeting the fornix by 133%. CONCLUSIONS: The described work allows for repeatable and accurate targeting of small and deep structures in the rodent brain, such as the fornix, enabling the investigation of neurological disorders in chronic disease models.

Development of an MR-guided focused ultrasound (MRgFUS) lesioning approach for small and deep structures in the rat brain.

Objective: High-intensity magnetic resonance-guided focused ultrasound (MRgFUS) is a noninvasive therapy to lesion brain tissue, used clinically in patients and preclinically in several animal models. Challenges with focused ablation in rodent brains can include skull and near-field heating and accurately targeting small and deep brain structures. We overcame these challenges by creating a novel method consisting of a craniectomy skull preparation, a high-frequency transducer (3 MHz) with a small ultrasound focal spot, a transducer positioning system with an added manual adjustment of ∼0.1 mm targeting accuracy, and MR acoustic radiation force imaging for confirmation of focal spot placement. Methods: The study consisted of two main parts. First, two skull preparation approaches were compared. A skull thinning approach (n=7 lesions) was compared to a craniectomy approach (n=22 lesions), which confirmed a craniectomy was necessary to decrease skull and near-field heating. Second, the two transducer positioning systems were compared with the fornix chosen as a subcortical ablation target. We evaluated the accuracy of targeting using a high-frequency transducer with a small ultrasound focal spot and MR acoustic radiation force imaging. Results: Comparing a motorized adjustment system (∼1 mm precision, n=17 lesions) to the motorized system with an added micromanipulator (∼0.1 mm precision, n=14 lesions), we saw an increase in the accuracy of targeting the fornix by 133%. The described work allows for repeatable and accurate targeting of small and deep structures in the rodent brain, such as the fornix, enabling the investigation of neurological disorders in chronic disease models.

Identification of clinically relevant biomarkers of epileptogenesis - a strategic roadmap.

Onset of many forms of epilepsy occurs after an initial epileptogenic insult or as a result of an identified genetic defect. Given that the precipitating insult is known, these epilepsies are, in principle, amenable to secondary prevention. However, development of preventive treatments is difficult because only a subset of individuals will develop epilepsy and we cannot currently predict which individuals are at the highest risk. Biomarkers that enable identification of these individuals would facilitate clinical trials of potential anti-epileptogenic treatments, but no such prognostic biomarkers currently exist. Several putative molecular, imaging, electroencephalographic and behavioural biomarkers of epileptogenesis have been identified, but clinical translation has been hampered by fragmented and poorly coordinated efforts, issues with inter-model reproducibility, study design and statistical approaches, and difficulties with validation in patients. These challenges demand a strategic roadmap to facilitate the identification, characterization and clinical validation of biomarkers for epileptogenesis. In this Review, we summarize the state of the art with respect to biomarker research in epileptogenesis and propose a five-phase roadmap, adapted from those developed for cancer and Alzheimer disease, that provides a conceptual structure for biomarker research.

Discovery of the First Vitamin K Analogue as a Potential Treatment of Pharmacoresistant Seizures.

Despite the availability of more than 25 antiseizure drugs on the market, approximately 30% of patients with epilepsy still suffer from seizures. Thus, the epilepsy therapy market has a great need for a breakthrough drug that will aid pharmacoresistant patients. In our previous study, we discovered a vitamin K analogue, 2h, which displayed modest antiseizure activity in zebrafish and mouse seizure models. However, there are limitations to this compound due to its pharmacokinetic profile. In this study, we develop a new series of vitamin K analogues by modifying the structure of 2h. Among these, compound 3d shows full protection in a rodent pharmacoresistant seizure model with limited rotarod motor toxicity and favorable pharmacokinetic properties. Furthermore, the brain/plasma concentration ratio of 3d indicates its excellent permeability into the brain. The resulting data shows that 3d can be further developed as a potential antiseizure drug in the clinic.

Genetic and pharmacological manipulation of glial glutamate transporters does not alter infection-induced seizure activity.

The contribution of glial transporters to glutamate movement across the membrane has been identified as a potential target for anti-seizure therapies. Two such glutamate transporters, GLT-1 and system xc-, are expressed on glial cells, and modulation of their expression and function have been identified as a means by which seizures, neuronal injury, and gliosis can be reduced in models of brain injury. While GLT-1 is responsible for the majority of glutamate uptake in the brain, system xc- releases glutamate in the extracellular cleft in exchange for cystine and represents as such the major source of hippocampal extracellular glutamate. Using the Theiler's Murine Encephalomyelitis Virus (TMEV) model of viral-induced epilepsy, we have taken two well-studied approaches, one pharmacological, one genetic, to investigate the potential role(s) of GLT-1 and system xc- in TMEV-induced pathology. Our findings suggest that the methods we utilized to modulate these glial transporters, while effective in other models, are not sufficient to reduce the number or severity of behavioral seizures in TMEV-infected mice. However, genetic knockout of xCT, the specific subunit of system xc-, may have cellular effects, as we observed a slight decrease in neuronal injury caused by TMEV and an increase in astrogliosis in the CA1 region of the hippocampus. Furthermore, xCT knockout caused an increase in GLT-1 expression selectively in the cortex. These findings have significant implications for both the characterization of the TMEV model as well as for future efforts to discover novel and effective anti-seizure drugs.

Hippocampal TNFα Signaling Contributes to Seizure Generation in an Infection-Induced Mouse Model of Limbic Epilepsy.

Central nervous system infection can induce epilepsy that is often refractory to established antiseizure drugs. Previous studies in the Theiler's murine encephalomyelitis virus (TMEV)-induced mouse model of limbic epilepsy have demonstrated the importance of inflammation, especially that mediated by tumor necrosis factor-α (TNFα), in the development of acute seizures. TNFα modulates glutamate receptor trafficking via TNF receptor 1 (TNFR1) to cause increased excitatory synaptic transmission. Therefore, we hypothesized that an increase in TNFα signaling after TMEV infection might contribute to acute seizures. We found a significant increase in both mRNA and protein levels of TNFα and the protein expression ratio of TNF receptors (TNFR1:TNFR2) in the hippocampus, a brain region most likely involved in seizure initiation, after TMEV infection, which suggests that TNFα signaling, predominantly through TNFR1, may contribute to limbic hyperexcitability. An increase in hippocampal cell-surface glutamate receptor expression was also observed during acute seizures. Although pharmacological inhibition of TNFR1-mediated signaling had no effect on acute seizures, several lines of genetically modified animals deficient in either TNFα or TNFRs had robust changes in seizure incidence and severity after TMEV infection. TNFR2-/- mice were highly susceptible to developing acute seizures, suggesting that TNFR2-mediated signaling may provide beneficial effects during the acute seizure period. Taken together, the present results suggest that inflammation in the hippocampus, caused predominantly by TNFα signaling, contributes to hyperexcitability and acute seizures after TMEV infection. Pharmacotherapies designed to suppress TNFR1-mediated or augment TNFR2-mediated effects of TNFα may provide antiseizure and disease-modifying effects after central nervous system infection.

Oxidative stress in murine Theiler's virus-induced temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of acquired epilepsy that can be caused by several inciting events including viral infections. However, one-third of TLE patients are pharmacoresistant to current antiepileptic drugs and therefore, there is an urgent need to develop antiepileptogenic therapies that prevent the development of the disease. Oxidative stress and redox alterations have recently been recognized as important etiological factors contributing to seizure-induced neuronal damage. The goal of this study was to determine if oxidative stress occurs in the TMEV (Theiler's murine encephalomyelitis virus) model of temporal lobe epilepsy (TLE). C57Bl/6 mice were injected with TMEV or with PBS intracortically and observed for acute seizures. At various time points after TMEV injection, hippocampi were analyzed for levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and 3-nitrotyrosine (3 NT). Mice infected with TMEV displayed behavioral seizures between days 3 and 7 days post-infection (dpi). The intensity of seizures increased over time with most of the seizures being a stage 4 or 5 on the Racine scale at 6 days p.i. Mice exhibiting at least one seizure during the observation period were utilized for the biochemical analyses. The levels of GSH were significantly depleted in TMEV infected mice at 3, 4 and 14 days p.i. with a concomitant increase in GSSH levels as well as an impairment of the redox status. Additionally, there was a substantial increase in 3 NT levels in TMEV infected mice at these time points. These redox changes correlated with the occurrence of acute seizures in this model. Interestingly, we did not see changes in any of the indices in the cerebellum of TMEV-infected mice at 3 dpi indicating that these alterations are localized to the hippocampus and perhaps other limbic regions. This is the first study to demonstrate the occurrence of oxidative stress in the TMEV model of infection-induced TLE. The redox alterations were observed at time points coinciding with the appearance of acute behavioral seizures suggesting that these changes might be a consequence of seizure activity. Our results support the hypothesis that redox changes correlate with seizure activity in acquired epilepsies, regardless of the inciting insults, and suggest oxidative stress as a potential therapeutic target for their treatment.

Once initiated, viral encephalitis-induced seizures are consistent no matter the treatment or lack of interleukin-6.

Theiler's murine encephalomyelitis virus infection of C57BL/6 mice results in acute behavioral seizures in 50% of the mice. Treatment of infected mice with minocycline or infection of interleukin (IL)-6-deficient chimeric mice results in a significant decrease in the number of mice developing seizures. However, in those mice that do develop seizures, the pathological changes (neuronal cell loss, inflammation [perivascular cuffing, gliosis, activated microglia/macrophages]), and the numbers of virus infected cells in minocycline-treated or IL-6-deficient chimeric mice are very similar. Therefore, once seizures develop, the pathological changes are consistent regardless of the treatment or genetic background.

cAMP-dependent protein kinase A activity modulates topiramate potentiation of GABA(A) receptors.

Activation of cAMP-dependent protein kinase A (PKA) prevents inhibition of non-NMDA glutamate receptors by the anticonvulsant topiramate. Using two-electrode voltage-clamp techniques, we demonstrate that PKA activity also modulates topiramate potentiation of recombinant GABA(A) receptors expressed in Xenpus laevis oocytes. PKA activators, dibutyryl-cAMP and forskolin, attenuate topiramate potentiation, whereas the PKA inhibitor H-89 increases topiramate potentiation. Thus, endogenous PKA activity and receptor phosphorylation states may contribute to topiramate treatment efficacy.

Interleukin-6, produced by resident cells of the central nervous system and infiltrating cells, contributes to the development of seizures following viral infection.

Cells that can participate in an innate immune response within the central nervous system (CNS) include infiltrating cells (polymorphonuclear leukocytes [PMNs], macrophages, and natural killer [NK] cells) and resident cells (microglia and sometimes astrocytes). The proinflammatory cytokine interleukin-6 (IL-6) is produced by all of these cells and has been implicated in the development of behavioral seizures in the Theiler's murine encephalomyelitis virus (TMEV)-induced seizure model. The assessment, via PCR arrays, of the mRNA expression levels of a large number of chemokines (ligands and receptors) in TMEV-infected and mock-infected C57BL/6 mice both with and without seizures did not clearly demonstrate the involvement of PMNs, monocytes/macrophages, or NK cells in the development of seizures, possibly due to overlapping function of the chemokines. Additionally, C57BL/6 mice unable to recruit or depleted of infiltrating PMNs and NK cells had seizure rates comparable to those of controls following TMEV infection, and therefore PMNs and NK cells do not significantly contribute to seizure development. In contrast, C57BL/6 mice treated with minocycline, which affects monocytes/macrophages, microglial cells, and PMNs, had significantly fewer seizures than controls following TMEV infection, indicating monocytes/macrophages and resident microglial cells are important in seizure development. Irradiated bone marrow chimeric mice that were either IL-6-deficient mice reconstituted with wild-type bone marrow cells or wild-type mice reconstituted with IL-6-deficient bone marrow cells developed significantly fewer behavioral seizures following TMEV infection. Therefore, both resident CNS cells and infiltrating cells are necessary for seizure development.

Innate but not adaptive immune responses contribute to behavioral seizures following viral infection.

PURPOSE: To examine the role of innate immunity in a novel viral infection-induced seizure model. METHODS: C57BL/6 mice, mouse strains deficient in interleukin (IL)-1RI, IL-6, tumor necrosis factor (TNF)-RI, or myeloid differentiation primary response gene 88 (MyD88), or transgenic mice (OT-I) were infected with Theiler's murine encephalomyelitis virus (TMEV) or were mock infected. Mice were followed for acute seizures. Tissues were examined for neuron loss, the presence of virus (viral RNA and antigen), perivascular cuffs, macrophages/microglia, and gliosis, and mRNA expression of IL-1, TNF-alpha, and IL-6. RESULTS: IL-1 does not play a major role in seizures, as IL-1RI- and MyD88-deficient mice displayed a comparable seizure frequency relative to controls. In contrast, TNF-alpha and IL-6 appear to be important in the development of seizures, as only 10% and 15% of TNF-RI- and IL-6-deficient mice, respectively, showed signs of seizure activity. TNF-alpha and IL-6 mRNA levels also increased in mice with seizures. Inflammation (perivascular cuffs, macrophages/microglia, and gliosis) was greater in mice with seizures. OT-I mice (virus persists) had a seizure rate that was comparable to controls (no viral persistence), thereby discounting a role for TMEV-specific T cells in seizures. DISCUSSION: We have implicated the innate immune response to viral infection, specifically TNF-alpha and IL-6, and concomitant inflammatory changes in the brain as contributing to the development of acute seizures. This model is a potential infection-driven model of mesial temporal lobe epilepsy with hippocampal sclerosis.

The effect of CGX-1007 and CI-1041, novel NMDA receptor antagonists, on NMDA receptor-mediated EPSCs.

The N-methyl-D-aspartate (NMDA) receptor-gated ion channel is comprised of at least one NR1 subunit and any of four NR2 subunits (NR2A-D). The NR2 subunit confers different pharmacological and kinetic properties to the receptor. CGX-1007 (Conantokin G), a 17-amino acid polypeptide isolated from the venom of Conus geographus, is a novel NMDA receptor antagonist that is thought to be selective for the NR2B subunit. CGX-1007 has been reported to have highly potent, broad-spectrum anticonvulsant activity in animal seizure models. CI-1041 is an investigational compound, which also possesses anticonvulsant activity and has been shown to be highly selective for NR2B containing NMDA receptors. Although both CI-1041 and CGX-1007 are reportedly NR2B specific antagonists, they differ in their ability to block amygdala-kindled seizures, suggesting that the mechanism of action of these compounds differs. The present study was designed to test the hypothesis that CI-1041 and CGX-1007 would differentially modulate the function of NMDA receptors at excitatory synapses. Using the whole cell patch clamp technique, CGX-1007 and CI-1041 were found to block CA1 pyramidal cell, NMDA receptor-mediated excitatory postsynaptic currents (N-EPSCs) in a concentration-dependent manner in hippocampal slices from P4-P6 animals. In contrast, only CGX-1007 decreased NMDA receptor-mediated EPSC peak amplitude in slices from adult animals. The CGX-1007 block of peak amplitude was accompanied by a similar concentration-dependent decrease in decay kinetics of NMDA receptor-mediated EPSCs. These results suggest that while CI-1041 may be selective for NMDA receptors containing the NR2B subunit, CGX-1007 appears to be less selective than previously reported.

Anticonvulsant profile and teratogenicity of N-methyl-tetramethylcyclopropyl carboxamide: a new antiepileptic drug.

PURPOSE: The studies presented here represent our efforts to investigate the anticonvulsant activity of N-methyl-tetramethylcyclopropyl carboxamide (M-TMCD) and its metabolite tetramethylcyclopropyl carboxamide (TMCD) in various animal (rodent) models of human epilepsy, and to evaluate their ability to induce neural tube defects (NTDs) and neurotoxicity. METHODS: The anticonvulsant activity of M-TMCD and TMCD was determined after intraperitoneal (i.p.) administration to CF#1 mice, and either oral or i.p. administration to Sprague-Dawley rats. The ability of M-TMCD and TMCD to block electrical-, chemical-, or sensory-induced seizures was examined in eight animal models of epilepsy. The plasma and brain concentrations of M-TMCD and TMCD were determined in the CF#1 mice after i.p. administration. The induction of NTDs by M-TMCD and TMCD was evaluated after a single i.p. administration at day 8.5 of gestation in a highly inbred mouse strain (SWV) that is susceptible to valproic acid-induced neural tube defects. RESULTS: In mice, M-TMCD afforded protection against maximal electroshock (MES)-induced, pentylenetetrazol (Metrazol)-induced, and bicuculline-induced seizures, as well as against 6-Hz "psychomotor" seizures and sound-induced seizures with ED50 values of 99, 39, 81, 51, and 10 mg/kg, respectively. In rats, M-TMCD effectively prevented MES- and Metrazol-induced seizures and secondarily generalized seizures in hippocampal kindled rats (ED50 values of 82, 45, and 39 mg/kg, respectively). Unlike M-TMCD, TMCD was active only against Metrazol-induced seizures in mice and rats (ED50 values of 57 and 52 mg/kg, respectively). Neither M-TMCD nor TMCD was found to induce NTDs in SWV mice. CONCLUSIONS: The results obtained in this study show that M-TMCD is a broad-spectrum anticonvulsant drug that does not induce NTDs and support additional studies to evaluate its full therapeutic potential.