Long-term outcome in a noninvasive rat model of birth asphyxia with neonatal seizures: Cognitive impairment, anxiety, epilepsy, and structural brain alterations.

OBJECTIVE: Birth asphyxia is a major cause of hypoxic-ischemic encephalopathy (HIE) in neonates and often associated with mortality, neonatal seizures, brain damage, and later life motor, cognitive, and behavioral impairments and epilepsy. Preclinical studies on rodent models are needed to develop more effective therapies for preventing HIE and its consequences. Thus far, the most popular rodent models have used either exposure of intact animals to hypoxia-only, or a combination of hypoxia and carotid occlusion, for the induction of neonatal seizures and adverse outcomes. However, such models lack systemic hypercapnia, which is a fundamental constituent of birth asphyxia with major effects on neuronal excitability. Here, we use a recently developed noninvasive rat model of birth asphyxia with subsequent neonatal seizures to study later life adverse outcome. METHODS: Intermittent asphyxia was induced for 30 min by exposing male and female postnatal day 11 rat pups to three 7 + 3-min cycles of 9% and 5% O2 at constant 20% CO2 . All pups exhibited convulsive seizures after asphyxia. A set of behavioral tests were performed systematically over 14 months following asphyxia, that is, a large part of the rat's life span. Video-electroencephalographic (EEG) monitoring was used to determine whether asphyxia led to the development of epilepsy. Finally, structural brain alterations were examined. RESULTS: The animals showed impaired spatial learning and memory and increased anxiety when tested at an age of 3-14 months. Video-EEG at ~10 months showed an abundance of spontaneous seizures, which was paralleled by neurodegeneration in the hippocampus and thalamus, and by aberrant mossy fiber sprouting. SIGNIFICANCE: The present model of birth asphyxia recapitulates several of the later life consequences associated with human HIE. This model thus allows evaluation of the efficacy of novel therapies designed to prevent HIE and seizures following asphyxia, and of how such therapies might alleviate long-term adverse consequences.

Systematic evaluation of rationally chosen multitargeted drug combinations: a combination of low doses of levetiracetam, atorvastatin and ceftriaxone exerts antiepileptogenic effects in a mouse model of acquired epilepsy.

Epileptogenesis, the gradual process that leads to epilepsy after brain injury or genetic mutations, is a complex network phenomenon, involving a variety of morphological, biochemical and functional brain alterations. Although risk factors for developing epilepsy are known, there is currently no treatment available to prevent epilepsy. We recently proposed a multitargeted, network-based approach to prevent epileptogenesis by rationally combining clinically available drugs and provided first proof-of-concept that this strategy is effective. Here we evaluated eight novel rationally chosen combinations of 14 drugs with mechanisms that target different epileptogenic processes. The combinations consisted of 2-4 different drugs per combination and were administered systemically over 5 days during the latent epileptogenic period in the intrahippocampal kainate mouse model of acquired temporal lobe epilepsy, starting 6 h after kainate. Doses and dosing intervals were based on previous pharmacokinetic and tolerability studies in mice. The incidence and frequency of spontaneous electrographic and electroclinical seizures were recorded by continuous (24/7) video linked EEG monitoring done for seven days at 4 and 12 weeks post-kainate, i.e., long after termination of drug treatment. Compared to vehicle controls, the most effective drug combination consisted of low doses of levetiracetam, atorvastatin and ceftriaxone, which markedly reduced the incidence of electrographic seizures (by 60%; p<0.05) and electroclinical seizures (by 100%; p<0.05) recorded at 12 weeks after kainate. This effect was lost when higher doses of the three drugs were administered, indicating a synergistic drug-drug interaction at the low doses. The potential mechanisms underlying this interaction are discussed. We have discovered a promising novel multitargeted combination treatment for modifying the development of acquired epilepsy.

Disruption of the sodium-dependent citrate transporter SLC13A5 in mice causes alterations in brain citrate levels and neuronal network excitability in the hippocampus.

In addition to tissues such as liver, the plasma membrane sodium-dependent citrate transporter, NaCT (SLC13A5), is highly expressed in brain neurons, but its function is not understood. Loss-of-function mutations in the human SLC13A5 gene have been associated with severe neonatal encephalopathy and pharmacoresistant seizures. The molecular mechanisms of these neurological alterations are not clear. We performed a detailed examination of a Slc13a5 deletion mouse model including video-EEG monitoring, behavioral tests, and electrophysiologic, proteomic, and metabolomic analyses of brain and cerebrospinal fluid. The experiments revealed an increased propensity for epileptic seizures, proepileptogenic neuronal excitability changes in the hippocampus, and significant citrate alterations in the CSF and brain tissue of Slc13a5 deficient mice, which may underlie the neurological abnormalities. These data demonstrate that SLC13A5 is involved in brain citrate regulation and suggest that abnormalities in this regulation can induce seizures. The present study is the first to (i) establish the Slc13a5-knockout mouse model as a helpful tool to study the neuronal functions of NaCT and characterize the molecular mechanisms by which functional deficiency of this citrate transporter causes epilepsy and impairs neuronal function; (ii) evaluate all hypotheses that have previously been suggested on theoretical grounds to explain the neurological phenotype of SLC13A5 mutations; and (iii) indicate that alterations in brain citrate levels result in neuronal network excitability and increased seizure propensity.

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.

Proof-of-concept that network pharmacology is effective to modify development of acquired temporal lobe epilepsy.

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.

Network pharmacology for antiepileptogenesis: Tolerability and neuroprotective effects of novel multitargeted combination treatments in nonepileptic vs. post-status epilepticus mice.

Network-based approaches in drug discovery comprise both development of novel drugs interacting with multiple targets and repositioning of drugs with known targets to form novel drug combinations that interact with cellular or molecular networks whose function is disturbed in a disease. Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed multitargeted, network-based approaches to prevent epileptogenesis by combinations of clinically available drugs chosen to impact diverse epileptogenic processes. In order to test this strategy preclinically, we developed a multiphase sequential study design for evaluating such drug combinations in rodents, derived from human clinical drug development phases. Because pharmacokinetics of such drugs are known, only the tolerability of novel drug combinations needs to be evaluated in Phase I in öhealthy" controls. In Phase IIa, tolerability is assessed following an epileptogenic brain insult, followed by antiepileptogenic efficacy testing in Phase IIb. Here, we report Phase I and Phase IIa evaluation of 7 new drug combinations in mice, using 10 drugs (levetiracetam, topiramate, gabapentin, deferoxamine, fingolimod, ceftriaxone, α-tocopherol, melatonin, celecoxib, atorvastatin) with diverse mechanisms thought to be important in epileptogenesis. Six of the 7 drug combinations were well tolerated in mice during prolonged treatment at the selected doses in both controls and during the latent phase following status epilepticus induced by intrahippocampal kainate. However, none of the combinations prevented hippocampal damage in response to kainate, most likely because treatment started only 16-18 h after kainate. This suggests that antiepileptogenic or disease-modifying treatment may need to start earlier after the brain insult. The present data provide a rich collection of tolerable, network-based combinatorial therapies as a basis for antiepileptogenic or disease-modifying efficacy testing.