Small-molecule caspase-1 inhibitor CZL80 terminates refractory status epilepticus via inhibition of glutamatergic transmission.

Status epilepticus (SE), a serious and often life-threatening medical emergency, is characterized by abnormally prolonged seizures. It is not effectively managed by present first-line anti-seizure medications and could readily develop into drug resistance without timely treatment. In this study, we highlight the therapeutic potential of CZL80, a small molecule that inhibits caspase-1, in SE termination and its related mechanisms. We found that delayed treatment of diazepam (0.5 h) easily induces resistance in kainic acid (KA)-induced SE. CZL80 dose-dependently terminated diazepam-resistant SE, extending the therapeutic time window to 3 h following SE, and also protected against neuronal damage. Interestingly, the effect of CZL80 on SE termination was model-dependent, as evidenced by ineffectiveness in the pilocarpine-induced SE. Further, we found that CZL80 did not terminate KA-induced SE in Caspase-1-/- mice but partially terminated SE in IL1R1-/- mice, suggesting the SE termination effect of CZL80 was dependent on the caspase-1, but not entirely through the downstream IL-1ß pathway. Furthermore, in vivo calcium fiber photometry revealed that CZL80 completely reversed the neuroinflammation-augmented glutamatergic transmission in SE. Together, our results demonstrate that caspase-1 inhibitor CZL80 terminates diazepam-resistant SE by blocking glutamatergic transmission. This may be of great therapeutic significance for the clinical treatment of refractory SE.

(+)-Borneol enantiomer ameliorates epileptic seizure via decreasing the excitability of glutamatergic transmission.

Epilepsy is one common brain disorder, which is not well controlled by current pharmacotherapy. In this study we characterized the therapeutic potential of borneol, a plant-derived bicyclic monoterpene compound, in the treatment of epilepsy and elucidated the underlying mechanisms. The anti-seizure potency and properties of borneol were assessed in both acute and chronic mouse epilepsy models. Administration of (+)-borneol (10, 30, 100 mg/kg, i.p.) dose-dependently attenuated acute epileptic seizure in maximal-electroshock seizure (MES) and pentylenetetrazol (PTZ)-induced seizure models without obvious side-effect on motor function. Meanwhile, (+)-borneol administration retarded kindling-induced epileptogenesis and relieved fully kindled seizures. Importantly, (+)-borneol administration also showed therapeutic potential in kainic acid-induced chronic spontaneous seizure model, which was considered as a drug-resistant model. We compared the anti-seizure efficacy of 3 borneol enantiomers in the acute seizure models, and found (+)-borneol being the most satisfying one with long-term anti-seizure effect. In electrophysiological study conducted in mouse brain slices containing the subiculum region, we revealed that borneol enantiomers displayed different anti-seizure mechanisms, (+)-borneol (10 µM) markedly suppressed the high frequency burst firing of subicular neurons and decreased glutamatergic synaptic transmission. In vivo calcium fiber photometry analysis further verified that administration of (+)-borneol (100 mg/kg) inhibited the enhanced glutamatergic synaptic transmission in epilepsy mice. We conclude that (+)-borneol displays broad-spectrum anti-seizure potential in different experimental models via decreasing the glutamatergic synaptic transmission without obvious side-effect, suggesting (+)-borneol as a promising anti-seizure compound for pharmacotherapy in epilepsy.

Inflachromene attenuates seizure severity in mouse epilepsy models via inhibiting HMGB1 translocation.

Epilepsy is not well controlled by current anti-seizure drugs (ASDs). High mobility group box 1 (HMGB1) is a DNA-binding protein in the nucleus regulating transcriptional activity and maintaining chromatin structure and DNA repair. In epileptic brains, HMGB1 is released by activated glia and neurons, interacting with various receptors like Toll-like receptor 4 (TLR4) and downstream glutamatergic NMDA receptor, thus enhancing neural excitability. But there is a lack of small-molecule drugs targeting the HMGB1-related pathways. In this study we evaluated the therapeutic potential of inflachromene (ICM), an HMGB-targeting small-molecule inhibitor, in mouse epilepsy models. Pentylenetetrazol-, kainic acid- and kindling-induced epilepsy models were established in mice. The mice were pre-treated with ICM (3, 10 mg/kg, i.p.). We showed that ICM pretreatment significantly reduced the severity of epileptic seizures in all the three epilepsy models. ICM (10 mg/kg) exerted the most apparent anti-seizure effect in kainic acid-induced epileptic status (SE) model. By immunohistochemical analysis of brain sections from kainic acid-induced SE mice, we found that kainic acid greatly enhanced HMGB1 translocation in the hippocampus, which was attenuated by ICM pretreatment in subregion- and cell type-dependent manners. Notably, in CA1 region, the seizure focus, ICM pretreatment mainly inhibited HMGB1 translocation in microglia. Furthermore, the anti-seizure effect of ICM was related to HMGB1 targeting, as pre-injection of anti-HMGB1 monoclonal antibody (5 mg/kg, i.p.) blocked the seizure-suppressing effect of ICM in kainic acid-induced SE model. In addition, ICM pretreatment significantly alleviated pyramidal neuronal loss and granule cell dispersion in kainic acid-induced SE model. These results demonstrate that ICM is an HMGB-targeting small molecule with anti-seizure potential, which may help develop a potential drug for treating epilepsy.

Chemogenetic inhibition of subicular seizure-activated neurons alleviates cognitive deficit in male mouse epilepsy model.

Cognitive deficit is a common comorbidity in temporal lobe epilepsy (TLE) and is not well controlled by current therapeutics. How epileptic seizure affects cognitive performance remains largely unclear. In this study we investigated the role of subicular seizure-activated neurons in cognitive impairment in TLE. A bipolar electrode was implanted into hippocampal CA3 in male mice for kindling stimulation and EEG recording; a special promoter with enhanced synaptic activity-responsive element (E-SARE) was used to label seizure-activated neurons in the subiculum; the activity of subicular seizure-activated neurons was manipulated using chemogenetic approach; cognitive function was assessed in object location memory (OLM) and novel object recognition (NOR) tasks. We showed that chemogenetic inhibition of subicular seizure-activated neurons (mainly CaMKIIα+ glutamatergic neurons) alleviated seizure generalization and improved cognitive performance, but inhibition of seizure-activated GABAergic interneurons had no effect on seizure and cognition. For comparison, inhibition of the whole subicular CaMKIIα+ neuron impaired cognitive function in naïve mice in basal condition. Notably, chemogenetic inhibition of subicular seizure-activated neurons enhanced the recruitment of cognition-responsive c-fos+ neurons via increasing neural excitability during cognition tasks. Our results demonstrate that subicular seizure-activated neurons contribute to cognitive impairment in TLE, suggesting seizure-activated neurons as the potential therapeutic target to alleviate cognitive impairment in TLE.

HMGB1 in the mPFC governs comorbid anxiety in neuropathic pain.

BACKGROUND: Whether neuroinflammation causes comorbid mood disorders in neuropathic pain remains elusive. Here we investigated the role of high mobility group box 1 protein (HMGB1), a proinflammatory cytokine, in the medial prefrontal cortex (mPFC) in anxiety comorbidity of neuropathic pain. METHODS: Neuropathic pain was induced by partial transection of the infraorbital nerve (p-IONX) or partial sciatic nerve ligation (PSL) in mice and evaluated by measuring nociceptive thresholds to mechanical and heat stimulation. Anxiety-like behaviors were assessed by elevated plus maze, light dark box and open field tests. Aversive or anti-aversive effect was detected by conditioned place preference test. Neuronal activity was evaluated by single-unit and patch clamp recordings. The contribution of mPFC pyramidal neurons to anxiety was further examined by selectively inhibiting them by optogenetics. HMGB1 expression was measured by immunohistochemistry and western blotting. Antagonism of HMGB1 was achieved by injecting anti-HMGB1 monoclonal antibody (mAb) intracerebrally or intraperitoneally. RESULTS: Anxiety-like behaviors were presented earlier after p-IONX than after PSL. HMGB1 expression was upregulated in the mPFC temporally in parallel to anxiety onset, rather than in other regions associated with anxiety. The upregulation of HMGB1 expression and its translocation from the nucleus to cytoplasm in the mPFC occurred predominantly in neurons and were accompanied with activation of microglia and astrocytes. Infusion of anti-HMGB1 mAb into the mPFC during the early and late phases after either p-IONX or PSL alleviated anxiety-like behaviors and aversion without changing pain sensitization, while local infusion of exogenous ds-HMGB1, the proinflammatory form of HMGB1, into the mPFC induced anxiety and aversion but not pain sensitization in naïve mice. In addition to reversing established pain sensitization and anxiety simultaneously, intraperitoneal injection of anti-HMGB1 mAb reduced HMGB1 upregulation and suppressed the hyperexcitability of layer 2/3 pyramidal neurons in the mPFC after p-IONX. Moreover, optogenetic inhibition of mPFC pyramidal neurons alleviated anxiety in p-IONX mice. CONCLUSION: These results demonstrate that HMGB1 in the mPFC drives and maintains anxiety comorbidity in neuropathic pain by increasing the excitability of layer 2/3 pyramidal neurons, and justify antagonism of HMGB1, e.g., neutralization by mAb, as a promising therapeutic strategy for neuropathic pain with anxiety comorbidity.

Long-term music adjuvant therapy enhances the efficacy of sub-dose antiepileptic drugs in temporal lobe epilepsy.

AIMS: Noninvasive music adjuvant therapy shows great potential in improving seizure control when combined with routine antiepileptic drugs. However, the diversity of previous music protocols has resulted in disparate outcomes. The optimized protocol and features for music adjuvant therapy are still not fully understood which limits its feasibility. METHODS: By applying different regimens of music therapy in various temporal lobe epilepsy models, we evaluated the effect of music in combination with sub-dose drugs on epileptic seizures to determine the optimized protocol. RESULTS: A subgroup of kindled mice that were responsive to music adjuvant therapy was screened. In those mice, sub-dose drugs which were noneffective on kindled seizures, alleviated seizure severity after 12 h/day Mozart K.448 for 14 days. Shorter durations of music therapy (2 and 6 h/day) were ineffective. Furthermore, only full-length Mozart K.448, not its episodes or other music varieties, was capable of enhancing the efficacy of sub-dose drugs. This music therapeutic effect was not due to increasing cerebral drug concentration, but instead was related with the modulation of seizure electroencephalogram (EEG) spectral powers in the hippocampus. CONCLUSION: These results indicate that long-term full-length Mozart K.448 could enhance the anti-seizure efficacy of sub-dose drugs and may be a promising noninvasive adjuvant therapy for temporal lobe epilepsy.

Pharmaco-genetic inhibition of pyramidal neurons retards hippocampal kindling-induced epileptogenesis.

AIMS: Pharmaco-genetics emerges as a new promising approach for epileptic seizures. Whether it can modulate epileptogenesis is still unknown. METHODS: Here, parvalbumin neurons and pyramidal neurons of the seizure focus were transfected with engineered excitatory Gq-coupled human muscarinic receptor hM3Dq and engineered inhibitory Gi-coupled human muscarinic receptor hM4Di, respectively. And their therapeutic value in mouse hippocampal kindling-induced epileptogenesis was tested. RESULTS: Pharmaco-genetic activating parvalbumin neurons limitedly retarded the progression of behavioral seizure stage and afterdischarge duration (ADD) during epileptogenesis induced by kindling. Activating parvalbumin neurons delayed seizure development only in the early stage, but accelerated it in late stages. On the contrary, pharmaco-genetic inhibiting pyramidal neurons robustly retarded the progression of seizure stages and ADDs, which greatly delayed seizure development in both early and late stages. Although both pharmaco-genetic therapeutics efficiently alleviated the severity of acute kindling-induced seizures, pharmaco-genetic inhibiting pyramidal neurons were able to reverse the enhanced synaptic plasticity during epileptogenesis, compared with that of pharmaco-genetic activating parvalbumin neurons. CONCLUSION: Our results demonstrated that pharmaco-genetic inhibiting pyramidal neurons retard hippocampal kindling-induced epileptogenesis and reverse the enhanced synaptic plasticity during epileptogenesis, compared with that of pharmaco-genetic activating parvalbumin neurons. It suggests that pharmaco-genetics targeting pyramidal neurons may be a promising treatment for epileptogenesis.

Parabrachial nucleus circuit governs neuropathic pain-like behavior.

The lateral parabrachial nucleus (LPBN) is known to relay noxious information to the amygdala for processing affective responses. However, it is unclear whether the LPBN actively processes neuropathic pain characterized by persistent hyperalgesia with aversive emotional responses. Here we report that neuropathic pain-like hypersensitivity induced by common peroneal nerve (CPN) ligation increases nociceptive stimulation-induced responses in glutamatergic LPBN neurons. Optogenetic activation of GABAergic LPBN neurons does not affect basal nociception, but alleviates neuropathic pain-like behavior. Optogenetic activation of glutamatergic or inhibition of GABAergic LPBN neurons induces neuropathic pain-like behavior in naïve mice. Inhibition of glutamatergic LPBN neurons alleviates both basal nociception and neuropathic pain-like hypersensitivity. Repetitive pharmacogenetic activation of glutamatergic or GABAergic LPBN neurons respectively mimics or prevents the development of CPN ligation-induced neuropathic pain-like hypersensitivity. These findings indicate that a delicate balance between excitatory and inhibitory LPBN neuronal activity governs the development and maintenance of neuropathic pain.

Correction to: Gender difference in acquired seizure susceptibility in adult rats after early complex febrile seizures.

In the original publication of the article, the representative EEG of female rat pups with FS in Figure 1 C and D was incorrectly intercepted from that of male rat pups. This correction does not affect the conclusions of the paper. Figure 1 has been corrected on the online PDF version and displayed below.

Protective effect of carnosine on febrile seizures in immature mice.

Febrile seizures (FSs) are the most common type of convulsions in childhood and complex FSs represent an increased risk for development of temporal lobe epilepsy. The aim of this study was to analyze the anticonvulsant effects of carnosine, an endogenous dipeptide composed of alanine and histidine, on hyperthermia induced seizure in immature mice. Injection of carnosine significantly increased the latency and decreased the duration of FSs in a dose-dependent manner. In addition, histidine had similar effects on FSs as carnosine. The protective effect of carnosine or histidine was completely abolished by α-fluoromethylhistidine (α-FMH), a selective and irreversible histidine decarboxylase inhibitor, or in histidine decarboxylase deficient (HDC-KO) mice. Peripheral carnosine administration increased the level of carnosine, histidine and histamine in the cortex and hippocampus of mice pups, but decreased glutamate contents in the cortex and hippocampus. These results indicate that carnosine can protect against FSs in mice pups through its conversion to histamine, suggesting that it may serve as an efficient anti-FSs drug in the future.

The Pro-inflammatory Cytokine Interleukin-1ß is a Key Regulatory Factor for the Postictal Suppression in Mice.

AIMS: The postictal suppression (PS) is a common and important period following an epileptic seizure but has not been well studied. This study was designed to determine whether interleukin-1ß (IL-1ß) is involved in the PS. METHODS: The effects of IL-1ß on the PS were tested in three independent seizure models induced by hippocampal kindling, maximal electroshock seizure (MES), and 4-aminopyridine, respectively. RESULTS: IL-1R1 knockout or IL-1RA enhanced the seizure refractory phenomenon without influencing the baseline seizure threshold in intermittent MES model. IL-1ß attenuated the seizure refractory phenomenon without affecting the severity of the preceding seizures in hippocampal kindling model, while IL-1RA enhanced it. Besides, IL-1ß reduced the postictal EEG suppression period, while IL-1RA prolonged it. And IL-1ß showed no further effect on the postictal EEG suppression and seizure refractory phenomenon in IL-1R1 knockout mice. In addition, 30 min after intrahippocampal injection of 4-aminopyridine, IL-1ß increased the incidence of SE, while IL-1RA prolonged the intervals between recurrent seizures. CONCLUSIONS: This study provides the first direct evidence that IL-1ß is key regulatory factor for the PS, and its receptor IL-1R1 may be a potential target for adjuvant treatment of postictal problems.

Gender difference in acquired seizure susceptibility in adult rats after early complex febrile seizures.

Gender differences are involved in many neurological disorders including epilepsy. However, little is known about the effect of gender difference on the risk of epilepsy in adults with a specific early pathological state such as complex febrile seizures (FSs) in infancy. Here we used a well-established complex FS model in rats and showed that: (1) the susceptibility to seizures induced by hyperthermia, pentylenetetrazol (PTZ), and maximal electroshock (MES) was similar in male and female rat pups, while males were more susceptible to PTZ- and MES-induced seizures than age-matched females in normal adult rats; (2) adult rats with complex FSs in infancy acquired higher seizure susceptibility than normal rats; importantly, female FS rats were more susceptible to PTZ and MES than male FS rats; and (3) the protein expression of interleukin-1ß, an inflammatory factor associated with seizure susceptibility, was higher in adult FS females than in males, which may reflect a gender-difference phenomenon of seizure susceptibility. Our results provide direct evidence that the acquired seizure susceptibility after complex FSs is gender-dependent.

Dysfunction of thermoregulation contributes to the generation of hyperthermia-induced seizures.

Febrile seizures (FS) are generally defined as seizures taking place during fever. Long-term prognosis, including development of epilepsy and malformation of cognitive function, has been demonstrated after infantile FS. However, the mechanism that triggers seizures in hyperthermic environment is still unclear. We here found that the body temperature of rat pups that experienced experimental FS was markedly decreased (∼28°C) after they were removed from the hyperthermic environment. Both the seizure generation and the temperature drop after seizure attack were abolished by either pre-treatment with chlorpromazine (CPZ), which impairs the thermoregulation, or by an electrolytic lesion of the preoptic area and anterior hypothalamus (PO/AH). However, the non-steroidal anti-inflammatory drug celecoxib did not affect the seizure incidence and the decrease in body temperature after seizure attack. In addition, pentobarbital prevented the generation of seizures, but did not reverse the decrease of body temperature after FS. Therefore, our work indicates that an over-regulation of body temperature occurs during hyperthermic environment, and that the dysfunction of thermoregulation in the PO/AH following hyperthermia contributes to the generation of FS.