Sex differences in physiological response to increased neuronal excitability in a knockin mouse model of pediatric epilepsy.

BACKGROUND: Epilepsy is a common neurological disease; however, few if any of the currently marketed antiseizure medications prevent or cure epilepsy. Discovery of pathological processes in the early stages of epileptogenesis has been challenging given the common use of preclinical models that induce seizures in physiologically normal animals. Moreover, despite known sex dimorphism in neurological diseases, females are rarely included in preclinical epilepsy models. METHODS: We characterized sex differences in mice carrying a pathogenic knockin variant (p.N1768D) in the Scn8a gene that causes spontaneous tonic-clonic seizures (TCs) at ∼3 months of age and found that heterozygous females are more resilient than males in mortality and morbidity. To investigate the cellular mechanisms that underlie female resilience, we utilized blood-brain barrier (BBB) and hippocampal transcriptomic analyses in heterozygous mice before seizure onset (pre-TC) and in mice that experienced ∼20 TCs (post-TC). RESULTS: In the pre-TC latent phase, both sexes exhibited leaky BBB; however, patterns of gene expression were sexually dimorphic. Females exhibited enhanced oxidative phosphorylation and protein biogenesis, while males activated gliosis and CREB signaling. After seizure onset (chronic phase), females exhibited a metabolic switch to lipid metabolism, while males exhibited increased gliosis and BBB dysfunction and a strong activation of neuroinflammatory pathways. CONCLUSION: The results underscore the central role of oxidative stress and BBB permeability in the early stages of epileptogenesis, as well as sex dimorphism in response to increasing neuronal hyperexcitability. Our results also highlight the need to include both sexes in preclinical studies to effectively translate results of drug efficacy studies.

Greater female than male resilience to mortality and morbidity in the Scn8a mouse model of pediatric epilepsy.

AIMS: Females and males of all ages are affected by epilepsy; however, unlike many clinical studies, most preclinical research has focused on males. Genetic variants in the voltage-gated sodium channel gene, SCN8A, are associated with a broad spectrum of neurological and epileptic syndromes. Here we investigate sex differences in the natural history of the Scn8a-N1768D knockin mouse model of pediatric epilepsy. METHODS: We utilize 24/7 video to monitor juveniles and adults of both sexes to investigate variability in seizure activity (e.g. onset and frequency), mortality and morbidity, response to cannabinoids, and mode of death. We also monitor sleep architecture using a noninvasive piezoelectric method in order to identify factors that influence seizure severity and outcome. RESULTS: Both sexes had nearly 100% penetrance in seizure onset and early mortality. However, adult heterozygous (D/+) females were more resilient as exhibited by the ability to tolerate more seizures over a longer lifespan. Homozygous (D/D) juveniles did not exhibit a sex difference in overall survival. Female estrus cycle was disrupted before seizure onset, while sleep was disrupted in both sexes in association with seizure onset. Females typically died while in convulsive status epilepticus; however, a high proportion of males died while not experiencing behavioral seizures. Only juvenile and adult males benefited from cannabinoid administration. CONCLUSIONS: These results support the hypothesis that factors associated with sexual differentiation play a role in the neurobiology of epilepsy and point to the importance of including both sexes in the design of studies to identify new epilepsy therapies.

Morphine-induced osteolysis and hypersensitivity is mediated through toll-like receptor-4 in a murine model of metastatic breast cancer.

ABSTRACT: The propensity for breast cancer to metastasize to bone is coupled to the most common complaint among breast cancer patients: bone pain. Classically, this type of pain is treated using escalating doses of opioids, which lack long-term efficacy due to analgesic tolerance, opioid-induced hypersensitivity, and have recently been linked to enhanced bone loss. To date, the molecular mechanisms underlying these adverse effects have not been fully explored. Using an immunocompetent murine model of metastatic breast cancer, we demonstrated that sustained morphine infusion induced a significant increase in osteolysis and hypersensitivity within the ipsilateral femur through the activation of toll-like receptor-4 (TLR4). Pharmacological blockade with TAK242 (resatorvid) as well as the use of a TLR4 genetic knockout ameliorated the chronic morphine-induced osteolysis and hypersensitivity. Genetic MOR knockout did not mitigate chronic morphine hypersensitivity or bone loss. In vitro studies using RAW264.7 murine macrophages precursor cells demonstrated morphine-enhanced osteoclastogenesis that was inhibited by the TLR4 antagonist. Together, these data indicate that morphine induces osteolysis and hypersensitivity that are mediated, in part, through a TLR4 receptor mechanism.

Anti-seizure effects of walnut peptides in mouse models of induced seizure: The involvement of GABA and nitric oxide pathways.

Epilepsy is one of the foremost medical disorders. Oxidative stress is a well-known mechanism in epileptogenesis, and many studies suggest that oxidative stress affects the onset and evolution of epilepsy. Here we evaluated the walnut peptide extracts' anti-seizure property in three different mouse seizure models including pentylenetetrazole-induced clonic seizure, chemical kindling, and maximal electroshock. Walnut peptides (20 mg/Kg) were administered by intraperitoneal (IP) injection of mice 60 min before seizure induction in the three models. To delineate the mechanisms of walnut peptides anti-seizure activity, we evaluated the impact of diazepam, flumazenil, and a NOS inhibitor on this activity. Intraperitoneal administration of walnut peptides significantly increased the seizure threshold. Our results also demonstrated that walnut peptides exert their anti-seizure properties through the modulation of benzodiazepine receptors. Thus, walnut peptides may be considered as a new anti-convulsion agent, which can reduce seizure occurrence and slow down seizure progression.

Nitric oxide and glutamate are contributors of anti-seizure activity of rubidium chloride: A comparison with lithium.

The neuro-protective effects of rubidium and lithium as alkali metals have been reported for different central nervous system dysfunctions including mania and depression. The aim of this study was evaluating as well as comparing the effects of rubidium chloride (RbCl) and lithium chloride (LiCl) on different seizures paradigms in mice and determining the involvement of NMDA receptors and nitrergic pathway. To assess the seizures threshold, animals received intravenous pentylenetetrazole (PTZ, 0.5%; 1 mL/min). Male NMRI mice (6-8 weeks) received intraperitoneal (i.p.) injections of different doses of RbCl and LiCl. Doses greater than 10 mg/kg of RbCl showed a significant anticonvulsant activity 60 min after administration; the anticonvulsant effects of LiCl was observed at the doses more than 5 mg/kg and after 30 min in PTZ-induced seizure threshold. But, RbCl (10, 20 mg/kg, i.p) or LiCl (5, 10 mg/kg, i.p) injection did not induce protection against maximal electroshock (MES) or intraperitoneal injection of PTZ lethal dose (80 mg/kg)-induced seizure models. Pre-treatment with L-NAME (non-selective nitric oxide synthase (NOS) inhibitor, 10 mg/kg; i.p.) and 7-nitroindazole (selective neuronal NOS inhibitor, 30 mg/kg; i.p.) enhanced the anticonvulsive effects of both RbCl (5 mg/kg, i.p.) and LiCl (1 mg/kg, i.p.) in PTZ-induced seizure threshold model. Injection of MK-801 (NMDA receptor antagonist, 0.05 mg/kg; i.p.) before RbCl (5 mg/kg, i.p.; P < 0.001) and LiCl (1 mg/kg, i.p.; P < 0.001) administration increased the anti-seizure activity. But, treatment with L-arginine (precursor of nitric oxide, 100 mg/kg; i.p.) decreased the seizure threshold of both RbCl (20 mg/kg, i.p.; P < 0.001) and LiCl (10 mg/kg, i.p.; P < 0.001). Measurement of nitrite levels in hippocampus of animals revealed a remarkable reduction after treatment with RbCl (20 mg/kg, i.p; P < 0.05) and LiCl (10 mg/kg, i.p; P < 0.01). To conclude, rubidium may protect central nervous system against seizures in PTZ-induced seizures threshold model through NMDA/nitrergic pathways with a similarity to lithium effects in mice.

Methadone's effects on pentylenetetrazole-induced seizure threshold in mice: NMDA/opioid receptors and nitric oxide signaling.

Methadone is a synthetic opioid used to treat opiate withdrawal and addiction. Studies have demonstrated the impact of methadone on seizure susceptibility. This study investigated the modulatory impacts of acute and subchronic (three times daily for 5 days) intraperitoneal methadone treatment on pentylenetetrazole-induced clonic seizure threshold (CST) in mice, as well as the involvement of the nitric oxide, N-methyl-d-aspartate (NMDA), and µ-opioid pathways. Acute administration of different doses of methadone (0.1, 0.3, 1, and 3 mg/kg) 45 min before CST significantly decreased the seizure threshold. Additionally, pretreatment with noneffective doses of an opioid receptor antagonist (naltrexone) and NMDA receptor antagonists (ketamine and MK-801) inhibited methadone's proconvulsive activity in the acute phase, while l-NAME (a nonspecific nitric oxide synthase (NOS) inhibitor) did not affect that activity. In the subchronic phase, methadone (3 mg/kg) demonstrated an anticonvulsive effect. Although subchronic pretreatment with noneffective doses of l-NAME and 7-nitroindazole (a specific neuronal NOS inhibitor) reversed methadone's anticonvulsive activity, aminoguanidine (a specific inducible NOS inhibitor), naltrexone, MK-801, and ketamine did not change methadone's anticonvulsive characteristic. Our results suggest that NMDA and µ-opioid receptors may be involved in methadone's proconvulsive activity in the acute phase, while methadone's anticonvulsive activity may be modulated by neuronal NOS in the subchronic phase.

The Possible Role of Nitric Oxide Pathway in Pentylenetetrazole Preconditioning Against Seizure in Mice.

Preconditioning is defined as an induction of adaptive response in organs against lethal stimulation provoked by subsequent mild sublethal stress. Several chemical agents have been demonstrated to cause brain tolerance through preconditioning. The aim of the present study is to test the hypothesis that preconditioning with pentylenetetrazole (PTZ) may have protective effect against seizure induced by i.v. infusion of PTZ. Mice were preconditioned by low-dose administration of PTZ (25 mg/kg) for 5 consecutive days, and the threshold of seizure elicited by i.v. infusion of PTZ was measured. To investigate the possible role of nitric oxide, NOS inhibitor enzymes, including L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) (10 mg/kg), aminoguanidine (AG) (50 mg/kg), 7-nitroindazole (7-NI) (15 mg/kg), and L-arginine (L-arg) (60 mg/kg), were administered concomitantly with PTZ in both acute and chronic phases. Determination of seizure threshold revealed significant enhancement after preconditioning with low dose of PTZ. While the protective effect of PTZ preconditioning was enhanced after the administration of L-arg, it was reversed following administration of L-NAME and 7NI, suggesting the involvement of nitric oxide pathway as an underlying mechanism of PTZ-induced preconditioning. Preconditioning with PTZ led to brain tolerance and adaptive response in animal model of PTZ-induced seizure. This effect is in part due to the involvement of nitric oxide pathway.