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Refractoriness to antiseizure medications is still a major concern in the pharmacotherapy of epilepsy. For this reason, we decided to evaluate the combination of levetiracetam and cannabidiol, administered at a subthreshold dose, to limit the possible adverse effects of this phytocannabinoid. We administered levetiracetam (300 mg/kg/day, via osmotic minipumps), cannabidiol (120 mg/kg/day, injected once a day subcutaneously), or their combination for one week in epileptic rats. Saline-treated epileptic rats were the control group. Animals were monitored with video electroencephalography the week before and after the treatment. No changes were found in the controls. Levetiracetam did not significantly reduce the total seizure number or the overall seizure duration. Still, the overall number of seizures ( p < 0.001, Duncan's new multiple range test) and their total duration ( p < 0.01) increased in the week following treatment withdrawal. Cannabidiol did not change seizures when administered as a single drug. Instead, levetiracetam combined with cannabidiol resulted in a significant reduction in the overall number and duration of seizures ( p < 0.05), when comparing values measured during treatment with both pre- and post-treatment values. These findings depended on changes in convulsive seizures, while non-convulsive seizures were stable. These results suggest that cannabidiol determined a remarkable potentiation of levetiracetam antiseizure effects at a subthreshold dose.

Produced by the mitochondria and endoplasmic reticulum, neurosteroids such as allopregnanolone are neuroprotective molecules that influence various neuronal functions and regulate neuroinflammation. They are reduced in neurodegenerative diseases, while in the Wobbler mouse model, allopregnanolone and its precursor progesterone showed protective effects on motor neuron degeneration. This single-center case-control study included 37 patients with amyotrophic lateral sclerosis (ALS) and 28 healthy controls. Cerebrospinal fluid (CSF) neurosteroid levels were quantified using liquid chromatography-electrospray tandem mass spectrometry and compared between the two cohorts. Neurosteroid concentrations have been correlated with neuroinflammation and neurodegeneration biomarkers detected through an automated immunoassay, along with disease features and progression. Pregnenolone, progesterone, allopregnanolone, pregnanolone, and testosterone levels were significantly lower in ALS patients' CSF compared to healthy controls. A significant inverse correlation was found between neurofilament and neurosteroid levels. Neurosteroid concentrations did not correlate with disease progression, phenotype, genotype, or survival prediction. Our study suggests the independence of the disease features and its progression, from the dysregulation of neurosteroids in ALS patients' CSF. This neurosteroid reduction may relate to disease pathogenesis or be a consequence of disease-related processes, warranting further research. The inverse correlation between neurosteroids and neurofilament levels may indicate a failure of compensatory neuroprotective mechanisms against neurodegeneration.

Neurosteroids are pleiotropic molecules involved in various neurodegenerative diseases with neuroinflammation. We assessed neurosteroids' serum levels in a cohort of Parkinson's Disease (PD) patients with heterozygous glucocerebrosidase (GBA) mutations (GBA-PD) compared with matched cohorts of consecutive non-mutated PD (NM-PD) patients and healthy subjects with (GBA-HC) and without (NM-HC) GBA mutations. A consecutive cohort of GBA-PD was paired for age, sex, disease duration, Hoehn and Yahr stage, and comorbidities with a cohort of consecutive NM-PD. Two cohorts of GBA-HC and HC were also considered. Clinical assessment included the Movement Disorder Society revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) and the Montreal Cognitive Assessment (MoCA). Serum samples were processed and analyzed by liquid chromatography coupled with the triple quadrupole mass spectrometry. Twenty-two GBA-PD (males: 11, age: 63.68), 22 NM-PD (males: 11, age: 63.05), 14 GBA-HC (males: 8; age: 49.36), and 15 HC (males: 4; age: 60.60) were studied. Compared to NM-PD, GBA-PD showed more hallucinations and psychosis ( p < 0.05, Fisher's exact test) and higher MDS-UPDRS part-II ( p < 0.05). Most of the serum neurosteroids were reduced in both GBA-PD and NM-PD compared to the respective control cohorts, except for 5α-dihydroprogesterone. Allopregnanolone was the only neurosteroid significantly lower ( p < 0.01, Dunn's test) in NM-PD compared to GBA-PD patients. Only in GBA-PD, allopregnanolone, and pregnanolone levels correlated (Spearman) with a more severe MDS-UPDRS part-III. Allopregnanolone levels also negatively correlated with MoCA scores, and pregnanolone levels correlated with more pronounced bradykinesia. This pilot study provides the first observation of changes in neurosteroid peripheral levels in GBA-PD. The involvement of the observed changes in the development of neuropsychological and motor symptoms of GBA-PD deserves further attention.

Trilostane is a 3β-hydroxysteroid dehydrogenase/Δ 5-4 isomerase inhibitor able to produce a manyfold increase in brain levels of various neurosteroids, including allopregnanolone. We previously found that treatment with trilostane can slow down epileptogenesis in the kainic acid (KA) model of temporal lobe epilepsy. It is unknown whether trilostane may have a similar effect on the progression of epilepsy severity, as observed in KA-treated rats. Consequently, we investigated the effects of trilostane (50 mg/kg/day, 1 week) in epileptic rats, given 64 days after KA administration. Seizures were monitored by video-electrocorticographic recordings before and during the treatment with trilostane or vehicle (sesame oil), and neurosteroid levels were measured in serum and cerebral tissue using liquid chromatography-electrospray tandem mass spectrometry after treatment. Pregnenolone sulfate, pregnenolone, progesterone, 5α-dihydroprogesterone, and allopregnanolone peripheral levels were massively increased by trilostane. With the only exception of hippocampal pregnenolone sulfate, the other neurosteroids augmented in both the neocortex and hippocampus. Only pregnanolone levels were not upregulated by trilostane. As expected, a significant increase in the seizure occurrence was observed in rats receiving the vehicle, but not in the trilostane group. This suggests that the increased availability of neurosteroids produced a disease-modifying effect in the brain of epileptic rats., (© 2024 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

In view of inevitable recurrences despite resection, glioblastoma (GB) is still an unmet clinical need. Dealing with the stromal-cell derived factor 1-alpha (SDF-1α)/CXCR4 axis as a hallmark of infiltrative GB tumors and with the resection cavity situation, the present study described the effects and relevance of a new engineered micro-nanostructured SF-HA-Hep aerogel sponges, made of silk fibroin (SF), hyaluronic acid (HA) and heparin (Hep) and loaded with SDF-1α, to interfere with the GB ecosystem and residual GB cells, attracting and confining them in a controlled area before elimination. 70 µm-pore sponges were designed as an implantable scaffold to trap GB cells. They presented shape memory and fit brain cavities. Histological results after implantation in brain immunocompetent Fischer rats revealed that SF-HA-Hep sponges are well tolerated for more than 3 months while moderately and reversibly colonized by immuno-inflammatory cells. The use of human U87MG GB cells overexpressing the CXCR4 receptor (U87MG-CXCR4+) and responding to SDF-1α allowed demonstrating directional GB cell attraction and colonization of the device in vitro and in vivo in orthotopic resection cavities in Nude rats. Not modifying global survival, aerogel sponge implantation strongly shaped U87MG-CXCR4+ tumors in cavities in contrast to random infiltrative growth in controls. Overall, those results support the interest of SF-HA-Hep sponges as modifiers of the GB ecosystem dynamics acting as "cell meeting rooms" and biocompatible niches whose properties deserve to be considered toward the development of new clinical procedures. STATEMENT OF SIGNIFICANCE: Brain tumor glioblastoma (GB) is one of the worst unmet clinical needs. To prevent the relapse in the resection cavity situation, new implantable biopolymer aerogel sponges loaded with a chemoattractant molecule were designed and preclinically tested as a prototype targeting the interaction between the initial tumor location and its attraction by the peritumoral environment. While not modifying global survival, biocompatible SDF1-loaded hyaluronic acid and silk fibroin sponges induce directional GB cell attraction and colonization in vitro and in rats in vivo. Interestingly, they strongly shaped GB tumors in contrast to random infiltrative growth in controls. These results provide original findings on application of exogenous engineered niches that shape tumors and serve as cell meeting rooms for further clinical developments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nanotechnology application in cancer treatment is promising and is likely to quickly spread worldwide in the near future. To date, most scientific studies on nanomaterial development have focused on deepening the attitudes of end users and experts, leaving clinical practice implications unexplored. Neuro-oncology might be a promising field for the application of nanotechnologies, especially for malignant brain tumors with a low-survival rate such as glioblastoma (GBM). As to improving patients' quality of life and life expectancy, innovative treatments are worth being explored. Indeed, it is important to explore clinicians' intention to use experimental technologies in clinical practice. In the present study, we conducted an exploratory review of the literature about healthcare workers' knowledge and personal opinions toward nanomedicine. Our search (i) gives evidence for disagreement between self-reported and factual knowledge about nanomedicine and (ii) suggests the internet and television as main sources of information about current trends in nanomedicine applications, over scientific journals and formal education. Current models of risk assessment suggest time-saving cognitive and affective shortcuts, i.e., heuristics support both laypeople and experts in the decision-making process under uncertainty, whereas they might be a source of error. Whether the knowledge is poor, heuristics are more likely to occur and thus clinicians' opinions and perspectives toward new technologies might be biased.

Objective: Epileptogenesis after status epilepticus (SE) has a faster onset in rats treated to reduce brain levels of the anticonvulsant neurosteroid allopregnanolone with the 5α-reductase inhibitor finasteride; however, it still has to be evaluated whether treatments aimed at increasing allopregnanolone levels could result in the opposite effect of delaying epileptogenesis. This possibility could be tested using the peripherally active inhibitor of 3β-hydroxysteroid dehydrogenase/Δ 5-4 isomerase trilostane, which has been shown repeatedly to increase allopregnanolone levels in the brain., Methods: Trilostane (50 mg/kg) was administered subcutaneously once daily for up to six consecutive days, starting 10 min after intraperitoneal administration of kainic acid (15 mg/kg). Seizures were evaluated by video-electrocorticographic recordings for 70 days maximum, and endogenous neurosteroid levels were assessed by liquid chromatography-electrospray tandem mass spectrometry. Immunohistochemical staining was performed to evaluate the presence of brain lesions., Results: Trilostane did not alter the latency of kainic acid-induced SE onset or its overall duration. When compared to the vehicle-treated group, rats receiving six daily trilostane injections presented a remarkable delay of the first spontaneous electrocorticographic seizure and subsequent tonic-clonic spontaneous recurrent seizures (SRSs). Conversely, rats treated with only the first trilostane injection during SE did not differ from vehicle-treated rats in developing the SRSs. Notably, trilostane did not modify neuronal cell densities or the overall damage in the hippocampus. In comparison to the vehicle group, repeated administration of trilostane significantly decreased the activated microglia morphology in the subiculum. As expected, allopregnanolone and other neurosteroid levels were remarkably increased in the hippocampus and neocortex of rats treated for 6 days with trilostane, but pregnanolone was barely detectable. Neurosteroids returned to basal levels after a week of trilostane washout., Significance: Overall, these results suggest that trilostane led to a remarkable increase in allopregnanolone brain levels, which was associated with protracted effects on epileptogenesis., (© 2023 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

We obtained evidence that mouse BV2 microglia synthesize neurosteroids dynamically to modify neurosteroid levels in response to oxidative damage caused by rotenone. Here, we evaluated whether neurosteroids could be produced and altered in response to rotenone by the human microglial clone 3 (HMC3) cell line. To this aim, HMC3 cultures were exposed to rotenone (100 nM) and neurosteroids were measured in the culture medium by liquid chromatography with tandem mass spectrometry. Microglia reactivity was evaluated by measuring interleukin 6 (IL-6) levels, whereas cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. After 24 h (h), rotenone increased IL-6 and reactive oxygen species levels by approximately +37% over the baseline, without affecting cell viability; however, microglia viability was significantly reduced at 48 h ( p < 0.01). These changes were accompanied by the downregulation of several neurosteroids, including pregnenolone, pregnenolone sulfate, 5α-dihydroprogesterone, and pregnanolone, except for allopregnanolone, which instead was remarkably increased ( p < 0.05). Interestingly, treatment with exogenous allopregnanolone (1 nM) efficiently prevented the reduction in HMC3 cell viability. In conclusion, this is the first evidence that human microglia can produce allopregnanolone and that this neurosteroid is increasingly released in response to oxidative stress, to tentatively support the microglia's survival.

Abrupt withdrawal from antiepileptic drugs is followed by increased occurrence of epileptic seizures, a phenomenon known as the "rebound effect". By stopping treatment with levetiracetam (LEV 300 mg/kg/day, n = 15; vs. saline, n = 15), we investigated the rebound effect in adult male Sprague-Dawley rats. LEV was continuously administered using osmotic minipumps, 7 weeks after the intraperitoneal administration of kainic acid (15 mg/kg). The effects of LEV were determined by comparing time intervals, treatments, and interactions between these main factors. Seizures were evaluated by video-electrocorticographic recordings and power band spectrum analysis. Furthermore, we assessed endogenous neurosteroid levels by liquid chromatography-electrospray-tandem mass spectrometry. LEV significantly reduced the percentage of rats experiencing seizures, reduced the seizure duration, and altered cerebral levels of neurosteroids. In the first week of LEV discontinuation, seizures increased abruptly up to 700% ( p = 0.002, Tukey's test). The power of delta band in the seizure postictal component was related to the seizure occurrence after LEV withdrawal ( r 2 = 0.73, p < 0.001). Notably, allopregnanolone hippocampal levels were positively related to the seizure occurrence ( r 2 = 0.51, p = 0.02) and to the power of delta band ( r 2 = 0.67, p = 0.004). These findings suggest a role for the seizure postictal component in the rebound effect, which involves an imbalance of hippocampal neurosteroid levels.

Background: Neurosteroids modulate epileptic activity by interacting with the γ-aminobutyric acid type A receptor, but their brain levels are still undetermined., Objectives: We aimed to establish neurosteroid levels in the neocortex and hippocampus by liquid chromatography/mass spectrometry in epileptic rats., Methods: Kainic acid-treated rats were continuously monitored up to 9 weeks to determine seizure frequency by video electrocorticography (n = 23) and compared to age-matched controls monitored in the same manner (n = 11)., Results: Decreased allopregnanolone (-50%; p < 0.05, Mann-Whitney test) and pregnanolone levels (-64%; p < 0.01) were found in the hippocampus, whereas pregnenolone sulfate, pregnenolone, progesterone, and 5α-dihydroprogesterone were nonsignificantly reduced. No changes were found in the neocortex. Moreover, allopregnanolone (but not pregnanolone) levels were positively correlated with seizure frequency (r2 = 0.4606, p < 0.01)., Conclusion: These findings indicate a selective reduction in hippocampal levels of 3α-reduced neurosteroids. This reduction was partially mitigated by seizures in the case of allopregnanolone., (© 2020 S. Karger AG, Basel.)

Neuroinflammation, whose distinctive sign is the activation of microglia, is supposed to play a key role in the development and progression of neurodegenerative diseases. The aim of this investigation was to determine levels of neurosteroids produced by resting and injured BV-2 microglial cells. BV-2 cells were exposed to increasing concentrations of rotenone to progressively reduce their viability by increasing reactive oxygen species (ROS) production. BV-2 cell viability was significantly reduced 24, 48 and 72 h after rotenone (50-1000 nM) exposure. Concomitantly, rotenone (50-100 nM) determined a dose-independent augmentation of ROS production. Then, BV-2 cells were exposed to a single, threshold dose of rotenone (75 nM) to evaluate the overtime release of neurosteroids. In particular, pregnenolone, pregnenolone sulfate, progesterone, 5α-dihydroprogesterone (5α-DHP), allopregnanolone, and pregnanolone, were quantified in the culture medium by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. BV-2 cells synthesized all the investigated neurosteroids and, after exposure to rotenone, 5αDHP and pregnanolone production was remarkably increased. In conclusion, we found that BV-2 cells not only synthesize several neurosteroids, but further increase this production following oxidative damage. Pregnanolone and 5α-DHP may play a role in modifying the progression of neuroinflammation in neurodegenerative diseases.

Neurosteroids can modulate γ-aminobutyric acid type A receptor-mediated inhibitory currents. Recently, we discovered that the neurosteroids progesterone, 5α-dihydroprogesterone, allopregnanolone, and pregnanolone are reduced in the cerebrospinal fluid of patients with status epilepticus (SE). However, it is undetermined whether neurosteroids influence SE. For this reason, first we evaluated whether the inhibitor of adrenocortical steroid production trilostane (50 mg/kg) could modify the levels of neurosteroids in the hippocampus and neocortex, and we found a remarkable increase in pregnenolone, progesterone, 5α-dihydroprogesterone, and allopregnanolone levels using liquid chromatography tandem mass spectrometry. Second, we characterized the dynamics of SE in the presence of the varied neurosteroidal milieu by a single intraperitoneal kainic acid (KA; 15 mg/kg) injection in trilostane-treated rats and their controls. Convulsions started in advance in the trilostane group, already appearing 90 minutes after the KA injection. In contrast to controls, convulsions prevalently developed as generalized seizures with loss of posture in the trilostane group. However, this effect was transient, and convulsions waned 2 hours before the control group. Moreover, electrocorticographic traces of convulsions were shorter in trilostane-treated rats, especially at the 180-minute (P < .001) and 210-minute (P < .01) time points. These findings indicate that endogenous neurosteroids remarkably modulate SE dynamics., (© 2020 International League Against Epilepsy.)

Background/aims: Status epilepticus (SE) might be followed by temporal lobe epilepsy (TLE), a common neurologic disorder characterized by spontaneous recurrent seizures (SRSs). However, the relationship between SE and TLE is still incompletely characterized. For this reason, in a model of TLE we evaluated the lesion extent and the onset of SRSs to determine if they were influenced by the SE dynamics., Methods: Sixty-two adult male Sprague-Dawley rats were implanted for video-electrocorticographic (v-ECoG) monitoring and intraperitoneally treated with saline or kainic acid (KA, 15 mg/kg) at 8 weeks of age. v-ECoG recordings were obtained during SE, in the following 9 weeks, and assessed by amplitude or power band spectrum. Rats were euthanized 3 or 64 days after SE to evaluate the lesion., Results: SE lasted about 10 h during which the mean duration of convulsive seizures (CSs) increased from 39 s, at 30 min, to 603 s at 4 h. The gamma power peaked 30 min after the SE onset and its peak was correlated (r²=0.13, p=0.042) with the overall SE duration. Subsequently, the gamma power was reduced under the baseline until the end of SE. The theta power increased at approximately 150% of basal levels 3 h after KA injection, but it went back to basal levels with the full development of CSs. Interestingly, the timing of the first SRS in chronic epilepsy was correlated with the latency to develop the first CS with loss of posture during SE (r²=0.60, p<0.001). Additionally, the overall duration of CSs observed during SE was related to the number of damaged brain regions (r²=0.60, p=0.005), but it did not influence the timing of the first SRS in chronic epilepsy., Conclusion: Overall, our results show that the onset of chronic epilepsy is modulated by SE dynamics, whereas brain damage is related to prolonged convulsions in SE., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

The ketogenic diet (KD) is a high-fat, low carbohydrate nutritional treatment adopted in several countries for refractory epilepsy. However, the use of KD is limited by adverse events including growth retardation. In a previous investigation, we demonstrated that ghrelin is reduced in children maintained on KD for 3 months. As ghrelin regulates growth hormone (GH) secretion, it can be hypothesized that growth retardation depends on the reduced ghrelin availability. To assess this hypothesis, in this study we evaluate ghrelin and growth during 1 year of KD. We examined a small cohort of 6 children (2 males and 4 females, age range 3-10.4 years) affected by refractory epilepsy, who received the KD as add-on treatment. All patients were on drug polytherapy. Endpoints of the study were: (i) ghrelin plasma levels at 0, 15, 30, 90, and 365 days from KD onset, (ii) growth, and (iii) seizure control by ketogenesis. Ghrelin levels were -53 and -47% of basal levels, respectively, at 90 and 365 days ( P < 0.05 for both). Mean height index z scores were reduced, but not significantly, by comparing basal values with those at the end of observation. Instead, body mass index z scores slightly increased. Ketosis induced by the KD was within 2-5 mmol/L and satisfactorily reduced the seizure frequency (>50%) in all patients. We show that ghrelin plasma levels are consistently reduced in children with refractory epilepsy and maintained on the KD. This change was associated with low growth indexes in the majority of patients.

Neurosteroids such as allopregnanolone may play a role in epilepsy as positive modulators of inhibitory currents mediated by γ-aminobutyric acid type A (GABA A ) receptor. Indeed, these molecules have been consistently shown to be anticonvulsants in animal models, but their role is still unclear in patients. For this reason, we investigated neurosteroids in the cerebrospinal fluid (CSF) of patients with status epilepticus (SE) by liquid chromatography tandem-mass spectrometry. Patients were retrospectively identified within subjects who received a lumbar puncture in the 2007-2017 period. Seventy-three patients (median age 65, ranging from 13 to 94 years; 67% women) with SE were evaluated. Controls (n = 52, median age 53, ranging from 16 to 93 years; 65% women) were patients presenting with symptoms for which a lumbar puncture was required by clinical guidelines, and who were negative at the end of the diagnostic work-up. Progesterone was 64% lower in patients with SE (p < 0.001). With respect to progesterone, upstream pregnenolone sulfate and pregnenolone did not change. Instead, downstream 5α-dihydroprogesterone, pregnanolone and allopregnanolone were, respectively, 49% (p < 0.001), 21% (p < 0.01) and 37% (p < 0.001) lower than in controls. Duration or type of SE, age and sex did not consistently affect CSF neurosteroid levels in the SE cohort. Instead, pregnenolone sulfate (Spearman's ρ = 0.4335, p < 0.01), allopregnanolone (ρ = 0.4121, p < 0.05) and pregnanolone (ρ = 0.592, p < 0.001) levels significantly increased by aging in controls. We conclude that neurosteroidogenesis is defective in patients with SE., (© 2018 International Society for Neurochemistry.)

Colloidal nanoplatelets (NPLs), owing to their efficient and narrow-band luminescence, are considered as promising candidates for solution-processable light-emitting diodes (LEDs) with ultrahigh color purity. To date, however, the record efficiencies of NPL-LEDs are significantly lower than those of more-investigated devices based on spherical nanocrystals. This is particularly true for red-emitting NPL-LEDs, the best-reported external quantum efficiency (EQE) of which is limited to 0.63% (EQE = 5% for green NPL-LEDs). Here, we address this issue by introducing a charge-regulating layer of a polar and polyelectrolytic polymer specifically engineered with complementary trimethylammonium and phosphonate functionalities that provide high solubility in orthogonal polar media with respect to the NPL active layer, compatibility with the metal cathode, and the ability to control electron injection through the formation of a polarized interface under bias. Through this synergic approach, we achieve EQE = 5.73% at 658 nm (color saturation 98%) in completely solution processed LEDs. Remarkably, exposure to air increases the EQE to 8.39%, exceeding the best reports of red NPL-LEDs by over 1 order of magnitude and setting a new global record for quantum-dot LEDs of any color embedding solution-deposited organic interlayers. Considering the emission quantum yield of the NPLs (40 ± 5%), this value corresponds to a near-unity internal quantum efficiency. Notably, our devices show exceptional operational stability for over 5 h of continuous drive in air with no encapsulation, thus confirming the potential of NPLs for efficient, high-stability, saturated LEDs.

The ketogenic diet (KD) is increasingly used to treat epilepsy refractory to antiepileptic drugs and other neurological disorders. In animal models, the KD was found to increase the threshold to seizures induced by different convulsive stimulations. However, in models in which suprathreshold stimuli were used, a paradoxical seizure worsening was consistently observed in KD-fed animals. To better define this phenomenon, we characterized the electrographic response to seizures induced in mice which were treated with the KD, and then corneally stimulated at 6-Hz in four different sessions. We also evaluated the electroencephalogram (EEG) in three patients in which the KD was associated with a paradoxical worsening of epileptic seizures. Although seizures were initially less severe, a remarkable prolongation of the electrographic response was observed in mice receiving the KD from the second session of 6-Hz corneal stimulation and onwards. The EEG was also markedly altered in the presence of progressive seizure aggravation observed in children treated with the KD, specifically one affected by Lennox-Gastaut syndrome and two by type I lissencephaly,. These results suggest that when seizures are induced or recur because of resistance to therapeutic interventions, the KD may change the EEG by potentiating the electrographic epileptic activity., Competing Interests: The authors declare no conflict of interest.

Ghrelin, des-acyl ghrelin and other related peptides possess anticonvulsant activities. Although ghrelin and cognate peptides were shown to physiologically regulate only the ghrelin receptor, some of them were pharmacologically proved to activate the peroxisome proliferator-activated receptor gamma (PPARγ) through stimulation of the scavenger receptor CD36 in macrophages. In our study, we challenged the hypothesis that PPARγ could be involved in the anticonvulsant effects of EP-80317, a ghrelin receptor antagonist. For this purpose, we used the PPARγ antagonist GW9662 to evaluate the modulation of EP-80317 anticonvulsant properties in two different models. Firstly, the anticonvulsant effects of EP-80317 were studied in rats treated with pilocarpine to induce status epilepticus (SE). Secondly, the anticonvulsant activity of EP-80317 was ascertained in the repeated 6-Hz corneal stimulation model in mice. Behavioral and video electrocorticographic (ECoG) analyses were performed in both models. We also characterized levels of immunoreactivity for PPARγ in the hippocampus of 6-Hz corneally stimulated mice. EP-80317 predictably antagonized seizures in both models. Pretreatment with GW9662 counteracted almost all EP-80317 effects both in mice and rats. Only the effects of EP-80317 on power spectra of ECoGs recorded during repeated 6-Hz corneal stimulation were practically unaffected by GW9662 administration. Moreover, GW9662 alone produced a decrease in the latency of tonic-clonic seizures and accelerated the onset of SE in rats. Finally, in the hippocampus of mice treated with EP-80317 we found increased levels of PPARγ immunoreactivity. Overall, these results support the hypothesis that PPARγ is able to modulate seizures and mediates the anticonvulsant effects of EP-80317.

Patients affected by protocadherin 19 (PCDH19)-female limited epilepsy (PCDH19-FE) present a remarkable reduction in allopregnanolone blood levels. However, no information is available on other neuroactive steroids and the steroidogenic response to hormonal stimulation. For this reason, we evaluated allopregnanolone, pregnanolone, and pregnenolone sulfate by liquid chromatographic procedures coupled with electrospray tandem mass spectrometry in 12 unrelated patients and 15 age-matched controls. We also tested cortisol, estradiol, progesterone, and 17OH-progesterone using standard immunoassays. Apart from estradiol and progesterone, all the considered hormones were evaluated in basal condition and after stimulation with adrenocorticotropic hormone (ACTH). A generalized decrease in blood levels of almost all measured neuroactive steroids was found. When considering sexual development, cortisol and pregnenolone sulfate basal levels were significantly reduced in postpubertal girls affected by PCDH19-FE. Of interest, ACTH administration did not recover pregnenolone sulfate serum levels but restored cortisol to control levels. In prepubertal girls with PCDH19-FE, by challenging adrenal function with ACTH we disclosed defects in the production of cortisol, pregnenolone sulfate, and 17OH-progesterone, which were not apparent in basal condition. These findings point to multiple defects in peripheral steroidogenesis associated with and potentially relevant to PCDH19-FE. Some of these defects could be addressed by stimulating adrenocortical activity., (Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.)

Neuroactive steroids are increasingly considered as relevant modulators of neuronal activity. Especially allopregnanolone (AP) and pregnenolone sulfate (PS) have been shown to possess, respectively, anticonvulsant or proconvulsant properties. In view of the potential role of these steroids, we aimed at evaluating AP and PS levels in cerebrospinal fluid (CSF) and blood samples obtained from patients with status epilepticus (SE). To this purpose, we enrolled 41 patients affected by SE and 41 subjects investigated for nonepileptic neurologic disorders. Liquid chromatographic procedures coupled with electrospray tandem mass spectrometry and routine laboratory investigations were performed. Significantly lower AP levels were found in the CSF of patients affected by SE (-30%; p < 0.05, Mann-Whitney test). Notably, AP was not detectable in 28 of 41 patients affected by SE (p < 0.01 vs. controls, Fisher's exact test). In serum, AP levels did not differ in the two considered groups. Conversely, PS was present at similar levels in the investigated groups. Finally, differences in AP levels could not be explained by a variation in CSF albumin content. These findings indicate that AP is defective in the CSF of patients affected by SE. This phenomenon was not dependent on carriers for steroids, such as albumin., (Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.)

The 6-Hz corneal stimulation test is used to screen novel antiepileptic molecules to overcome the problem of drug refractoriness. Although recognized as a standard test, it has been evaluated only recently in the attempt to characterize the putative neuronal networks involved in seizures caused by corneal stimulation. In particular, by recording from the CA1 region we previously established that the hippocampus participates to propagation of seizure activity. However, these findings were not corroborated by using markers of neuronal activation such as FosB/ΔFosB antigens. In view of this discrepancy, we performed new experiments to characterize the changes in levels of phosphorylated extracellular signal-regulated kinases1/2 (p-ERK1/2), which are also used as markers of neuronal activation. To this aim, mice underwent corneal stimulation up to three different times, in three sessions separated by an interval of 3 days. To characterize a group in which seizures could be prevented by pharmacological treatment, we also considered pretreatment with the ghrelin receptor antagonist EP-80317 (330 μg/kg). Control mice were sham-treated. Video electrocorticographic (ECoG) recordings were obtained from mice belonging to each group of treatment. Animals were finally used to characterize the immunoreactivity for FosB/ΔFosB and p-ERK1/2 in the hippocampus. As previously shown, FosB/ΔFosB levels were highly increased throughout the hippocampus by the first induced seizure but, in spite of the progressively increased seizure severity, they were restored to control levels after the third stimulation. At variance, corneal stimulation caused a progressive increase in p-ERK1/2 immunoreactivity all over the hippocampus, especially in CA1, peaking in the third session. Predictably, EP-80317 administration reduced both duration and severity of seizures, prevented the increase in FosB/ΔFosB levels in the first session, and partially counteracted the increase in p-ERK1/2 levels in the third session. The vast majority of p-ERK1/2 immunopositive cells were co-labeled with FosB/ΔFosB antibodies, suggesting the existence of a relationship between the investigated markers in a subpopulation of neurons activated by seizures. These findings suggest that p-ERK1/2 are useful markers to define the aggravation of seizures and the response to anticonvulsant treatments. In particular, p-ERK1/2 expression clearly identified the involvement of hippocampal regions during seizure aggravation in the 6-Hz model.

Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca(2+) influx and mobilization of Ca(2+) from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51kDa and 14kDa subunits) or the Ca(2+)-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca(2+) response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca(2+) signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca(2+) oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca(2+) further supported the involvement of these organelles in staurosporine-induced Ca(2+) signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca(2+) dynamics and bioenergetics., (Copyright © 2015 Elsevier B.V. All rights reserved.)