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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.)

Status epilepticus (SE) is one of the recognized primary precipitating events that can lead to temporal lobe epilepsy (TLE) associated with hippocampal sclerosis. This type of epilepsy is characterized by poor response to drug treatment, often requiring surgical intervention to remove the mesial temporal regions involved in the seizure onset. However, even neurosurgery may not be completely successful. Thus, the prevention of hippocampal damage and epileptogenesis is currently evaluated as a possible alternative therapeutic approach to prevent the development of pharmacoresistant TLE. Lines of evidence suggest that ischemic-hypoxic lesions might occur in different brain regions, including the hippocampus, during SE. Especially in the hippocampal CA3 region, an ischemic-like lesion develops in the stratum lacunosum-moleculare and is mainly characterized by a loss of astrocytes and neuronal processes and increased immunostaining of pimonidazole which probes areas exposed to hypoxia. Interestingly, these mechanisms can contribute to neuronal cell loss and may be counteracted by drugs that can afford vascular protection, as in the case of ligands of the ghrelin receptor. Notably, some of the ghrelin receptor ligands possess a double edge effect, since they are anticonvulsant and vascular-protective, thus, potentially representing new tools to counteract the consequences of SE. This article is part of a Special Issue entitled "Status Epilepticus"., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Neurosteroids (NSs) are well known modulators of neuronal activity and by binding to different neuronal receptors are responsible for a broad spectrum of biological and pathophysiological conditions. Here, a sensitive liquid chromatographic-electrospray ionization-tandem mass spectrometric method (LC-ESI-MS/MS) has been developed and validated for the simultaneous determination in rat brain areas of three NSs, i.e. pregnenolone sulphate (PS), dehydroepiandrosterone (DHEA) and allopregnanolone (AP). NSs were extracted with methanol-formic acid, purified by Hybrid-SPE cartridges and subjected to LC-ESI-MS/MS without any preliminary derivatization or deconjugation procedure. Quantitation was performed by multiple reaction monitoring mode with the internal standard method, using deuterium-labelled analogues of the analyzed NSs. The proposed method provided for the first time a direct quantitative determination of PS without hydrolysis; in particular, PS concentrations were found significantly (p<0.01) higher in hippocampus, the brain area associated primarily with memory, than in cortical tissue of control rats, suggesting the important role of this NS in the process of memory formation. The developed method could be successfully applied to quantify simultaneously PS, DHEA and AP levels in brain tissue in order to study their changes during various neurodegenerative diseases and to investigate the role of PS in the brain., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Trilostane is a 3β‐hydroxysteroid dehydrogenase/Δ5‐4 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 kinases½ (p-ERK½), 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-ERK½ 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-ERK½ 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-ERK½ levels in the third session. The vast majority of p-ERK½ 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-ERK½ are useful markers to define the aggravation of seizures and the response to anticonvulsant treatments. In particular, p-ERK½ expression clearly identified the involvement of hippocampal regions during seizure aggravation in the 6-Hz model. [ABSTRACT FROM AUTHOR]