Mechanisms of the Cav2.3 Calcium Channel’s Role in Epileptogenesis and Antiepileptic Pharmacotherapy

RATIONALE: The Cav2.3 (R-type) voltage-gated calcium channel represents the most enigmatic of all voltage-gated calcium channels due to its pharmacoresistance, mixed characteristics of high voltage-activated and low voltage-activated calcium channels and relatively low expression levels. Lamotrigine (LTG) is a modern antiepileptic drug however, its mechanism of action has yet to be fully understood, as it is known to modulate several ion channels and other targets. In heterologous systems, LTG inhibits Cav2.3 (R-type) calcium currents, which contribute to kainic-acid (KA)–induced epilepsy in vivo. LTG has been suggested to increase the risk of sudden unexpected death in epilepsy (SUDEP), in which cardiac and respiratory mechanisms are proposed to be involved. In addition to the higher risk of SUDEP during sleep, epileptic patients are at higher risk of seizures during sleep, especially during slow wave sleep (SWS). The bidirectional relationship between sleep and epilepsy has long been acknowledged, however it remains far from understood. AIM: The goal of the present project was to perform an in depth investigation of the role of R-type signaling in the epileptic brain and heart, by analyzing its contribution to experimental epilepsy, antiepileptic pharmacotherapy and sleep. METHODS: In the first study we compared the effects of LTG to two other AEDs (Topiramate and Lacosamide) in Cav2.3-deficient mice and controls on KA-induced seizures. Behavioral seizure rating and quantitative electrocorticography were performed after KA induced epilepsy, as well as immunohistochemistry and western blot analysis of Cav2.3 expression in the brain. In the second study we investigated cardiac parameters during KA-induced epilepsy and LTG treatment in awake and sleeping C57Bl6 mice. Continuous electrocardiograms and electrocorticograms were collected telemetrically from freely moving mice, and time- and frequency-domain analysis performed on the electrocardiograms. In the third study, we analyzed sleep architecture in Cav2.3-deficient and control mice also using radiotelemetric electrocorticography and electromyography during spontaneous and urethane-induced sleep. RESULTS: LTG treatment displayed no antiepileptic potency in Cav2.3-deficient mice, but contrarily significantly aggravated seizures and increased neurodegeneration in the CA1 region of the hippocampus as well as increasing ultra-high frequency oscillations (ripples) known to be associated with seizure generation. This effect was specific to LTG in Cav2.3-deficient mice, as the two other AEDs tested - one with and one without Cav2.3 inhibiting capacity- did not aggravate seizures. In our second study we found LTG to alter autonomous nervous control of the heart during SWS after induction of chronic epilepsy promoting sympatho-vagal imbalance. Furthermore, we found LTG to increase the squared-coefficient of variation of the heart rate during SWS, but not during wakefulness. Our third study was able to demonstrate, that ablation of Cav2.3 robustly impacts sleep architecture, producing deficits in the amount and depth of SWS. Interestingly, although Cav2.3 mice sleep less and display shorter SWS phases, they do not compensate for this deficit by increasing sleep depth, pointing to disturbances in sleep homeostasis. DISCUSSION: We provide first in vivo evidence for a crucial role of R-type signaling in LTG pharmacology and shed light on a paradoxical effect of LTG in the absence of Cav2.3. LTG appears to promote ictal activity in Cav2.3-deficient mice by increasing high frequency components of seizures, resulting in increased neurotoxicity in the CA1. This paradoxical mechanism, possibly reflecting rebound hyperexcitation may be key in understanding LTG-induced seizure aggravation observed in patients. Furthermore, we find Cav2.3 to be a critical mediator of sleep homeostasis, potentially representing a pivotal link between sleep and epilepsy. Cav2.3 has been shown to be crucial for bursting in the reticular thalamus, which underlies delta-rhythm during SWS and generation of spike-and-wave discharges, the hallmark of absence epilepsy. Therefore, seizure resistance and SWS impairment of Cav2.3-deficient mice may be symptomatic of impairment of bursting in the thalamus and therefore of the generation and maintenance of highly synchronized slow rhythms. Remarkably, we found LTG to only affect autonomous control of the epileptic heart during SWS, possibly indicating a mechanism by which LTG could increase the risk for SUDEP. LTG-induced increased sympathetic tone during SWS, may also reflect impaired SWS, found in LTG-treated patients and in our Cav2.3-deficient mice. CONCLUSION: Because Cav2.3-deficient mice display a subtle phenotype as oppposed to an obvious one, because of the expression of Cav2.3 in rhythmically active tissue and because of Cav2.3’s unique electrophysiological properties, it is conceivable that a general function of R-type currents is the “fine tuning” of oscillatory networks One may assume that a loss of “fine-tuning” in Cav2.3KO mice is only minimally noticeable under physiological conditions, but becomes evident in certain pathological conditions exerting a strain on an oscillatory network such as during experimentally induced epilepsy. This may explain how R-type signaling is crucial for sustaining physiological rhythmic activity of an entire network despite relatively low expression levels

Vagus nerve stimulation in refractory and super-refractory status epilepticus - A systematic review.

Abstract Rationale Refractory status epilepticus (RSE) is the persistence of status epilepticus despite second-line treatment. Super-refractory SE (SRSE) is characterized by ongoing status despite 48 h of anaesthetic treatment. Due to the high case fatality in RSE of 16–39%, off label treatments without strong evidence of efficacy in RSE are often administered. In single case-reports and small case series totalling 28 patients, acute implantation of VNS in RSE was associated with 76% and 26% success rate in generalized and focal RSE respectively. We performed an updated systematic review of the literature on efficacy of VNS in RSE/SRSE by including all reported patients. Methods We systematically searched EMBASE, CENTRAL, Opengre.eu, and ClinicalTrials.gov , and PubMed databases to identify studies reporting the use of VNS for RSE and/or SRSE. We also searched conference abstracts from AES and ILAE meetings. Results 45 patients were identified in total of which 38 were acute implantations of VNS in RSE/SRSE. Five cases had VNS implantation for epilepsia partialis continua, one for refractory electrical status epilepticus in sleep and one for acute encephalitis with refractory repetitive focal seizures. Acute VNS implantation was associated with cessation of RSE/SRSE in 74% (28/38) of acute cases. Cessation did not occur in 18% (7/38) of cases and four deaths were reported (11%); all of them due to the underlying disease and unlikely related to VNS implantation. Median duration of the RSE/SRSE episode pre and post VNS implantation was 18 days (range: 3–1680 days) and 8 days (range: 3–84 days) respectively. Positive outcomes occurred in 82% (31/38) of cases. Conclusion VNS can interrupt RSE and SRSE in 74% of patients; data originate from reported studies classified as level IV and the risk for reporting bias is high. Further prospective studies are warranted to investigate acute VNS in RSE and SRSE

Can the combination of hyperthermia, seizures and ion channel dysfunction cause fatal post-ictal cerebral edema in patients with SCN1A mutations?

A 21-year-old male with an SCN1A mutation died of cerebral herniation 3 h after a seizure occurring during physical activity. Cases of fatal cerebral edema in patients with SCN1A mutations after fever and status epilepticus have been recently reported raising the question whether sodium channel dysfunction may contribute to cerebral edema and thereby contribute to the increased premature mortality in Dravet Syndrome. We report on our patient and discuss whether the combination of hyperthermia and ion channel dysfunction may not only trigger seizures but also a fatal pathophysiological cascade of cerebral edema and herniation leading to cardiorespiratory collapse. Keywords: Dravet Syndrome, SCN1A mutation, Fatality, Cerebral edema, Hyperthermia, SUDE

Cav2.3 (R-Type) Calcium Channels are Critical for Mediating Anticonvulsive and Neuroprotective Properties of Lamotrigine In Vivo

Background/Aims: Lamotrigine (LTG) is a popular modern antiepileptic drug (AED), however, its mechanism of action has yet to be fully understood, as it is known to modulate many members of several ion channel families. In heterologous systems, LTG inhibits Cav2.3 (R-type) calcium currents, which contribute to kainic-acid- (KA) induced epilepsy in vivo. To gain insight into the role of R-type currents in LTG drug action in vivo, we compared the effects of LTG to topiramate and lacosamide in Cav2.3-deficient mice and controls on KA-induced seizures. Methods: Behavioral seizure rating and quantitative electrocorticography were performed after injection of 20 mg/kg [and 30 mg/kg] KA. One hour before KA injection, mice were pretreated with either 30 mg/kg LTG, 50 mg/kg topiramate (TPM) or 30 mg/kg lacosamide (LSM). Results: Ablation of Cav2.3 reduced total seizure scores by 28.6% (p=0.0012) and pretreatment with LTG reduced seizure activity of control mice by 23.2% (p=0.02). In Cav2.3-deficient mice LTG pretreatment increased seizure activity by 22.1% (p=0.018) and increased the percentage of degenerated CA1 pyramidal neurons (p=0.02). All three tested AEDs reduced seizure activity in control mice, however only the non-calcium channel modulating AED, LSM had an anticonvulsive effect in Cav2.3-deficient mice. Furthermore LTG altered electrocorticographic parameters differently in the two genotypes, decreasing relative power of ictal spikes in control mice compared to Cav2.3-defcient mice. Conclusion: These findings give first in vivo evidence for an essential role for Cav2.3 in LTG pharmacology and shed light on a paradoxical effect of LTG in their absence. Furthermore, LTG appears to promote ictal activity in Cav2.3-deficient mice resulting in increased neurotoxicity in the CA1 region. This paradoxical mechanism, possibly reflecting rebound hyperexcitation of pyramidal CA1 neurons after increased inhibition, may be key in understanding LTG-induced seizure aggravation, observed in clinical practice

Aminolevulinic Acid-Mediated Photodynamic Therapy of Human Meningioma: An in Vitro Study on Primary Cell Lines

Objective: Five-aminolevulinic acid (5-ALA)-induced porphyrins in malignant gliomas are potent photosensitizers. Promising results of ALA-PDT (photodynamic therapy) in recurrent glioblastomas have been published. Recently, 5-ALA-induced fluorescence was studied in meningioma surgery. Here, we present an experimental study of ALA-PDT in an in vitro model of primary meningioma cell lines. Methods: We processed native tumor material obtained intra-operatively within 24 h for cell culture. Epithelial membrane antigen (EMA) immunohistochemistry was performed after the first passage to confirm that cells were meningioma cells. For 5-ALA-PDT treatment, about 5000 cells per well were seeded in 20 wells of a blank 96-well plate. Each block of 4 wells was inoculated with 150 µL of 0, 25, 50 and 100 µg/mL 5-ALA solutions; one block was used as negative control without 5-ALA and without PDT. Following incubation for 3 h PDT was performed using a laser (635 nm, 18.75 J/cm2). The therapeutic response was analyzed by the water soluble tetrazolium salt (WST-1) cell viability assay 90 min after PDT. Results: 5-ALA-PDT was performed in 14 primary meningioma cell lines. EMA expression was verified in 10 primary cell cultures. The remaining 4 were EMA negative and PDT was without any effect in these cultures. All 10 EMA-positive cell lines showed a significant and dose-dependent decrease in viability rate (p < 0.001). Cell survival at 5-ALA concentrations of 12.5, 25, 50 and 100 μg/mL was 96.5% ± 7.6%, 67.9% ± 29.9%, 24.0% ± 16.7% and 13.8% ± 7.5%, respectively. For the negative controls (no 5-ALA/PDT and ALA/no PDT), the viability rates were 101.72% ± 3.5% and 100.17% ± 3.6%, respectively. The LD50 for 5-ALA was estimated between 25 and 50 µg/mL. Conclusion: This study reveals dose-dependent cytotoxic effects of 5-ALA-PDT on primary cell lines of meningiomas. Either 5-ALA or PDT alone did not affect cell survival. Further efforts are necessary to study the potential therapeutic effects of 5-ALA-PDT in vivo

Cavernous brain malformations and their relation to black blood MRI in respect to vessel wall contrast enhancement

Abstract Background Inflammatory responses are implicated as crucial patho-mechanisms of vascular brain malformations. Inflammation is suggested to be a key contributor to aneurysm rupture; however it is unclear whether inflammation contributes similarly to bleeding of cerebral cavernous malformations (CCMs). Black blood MRI is a sequence which identifies inflammation in blood vessel walls and in the present study is used to detect inflammatory response in CCMs. Methods Fifteen patients with 17 CCMs treated in our department in 2017 were retrospectively analysed. All patients received black blood MRIs and the results were analysed in correlation with, size and bleeding of CCMs. Results Size and bleeding status of CCMs did not correlate with contrast enhancement in the CCM wall. One of 3 patients with bleeding displayed contrast enhancement in black blood MRI, whereas the others had non enhancing lesions. Because of the small number of cases a statistical analysis was not performed. Conclusion In this limited cohort, inflammatory reactions in CCMs could not be detected by black blood MRI suggesting that the level of inflammation is minimal in these lesions and those different patho-mechanisms play a more important role in the rupture of CCMs