Grygoriy Tsenov

Pregnanolone glutamate (PG) is a synthetic neurosteroid analog showing promise for treating ischemic brain injury, yet its blood–brain barrier (BBB) transport and metabolic fate remain unclear. We investigated the pharmacokinetics of PG in postnatal day 12 rats of both sexes subjected to endothelin-1 (ET-1)-induced focal hippocampal ischemia. Animals received PG (1 mg/kg intraperitoneal (i.p.)) or vehicle; serum and hippocampal steroidomes were profiled 60 min post-administration using gas chromatography-tandem mass spectrometry (GC-MS/MS) (hippocampus: n = 16 PG+, n = 27 PG−; multi-tissue subset: n = 6 PG+, n = 21 PG−). Our data revealed a “dual-fate” mechanism: PG undergoes systemic hydrolysis as a prodrug, as suggested by the tissue distribution pattern at 60 min post-administration, but also crosses the BBB intact, with significant parent conjugate accumulation in the hippocampus (42.3 pmol/g). The brain functioned as a “metabolic sink”, passively accumulating metabolites generated in peripheral organs—such as 17-hydroxypregnanolone—despite local absence of synthesizing enzymes (e.g., CYP17A1). Crucially, PG induced “metabolic segregation” within the central nervous system (CNS): the pharmacological 5β-pathway was saturated (~170-fold pregnanolone increase), while endogenous neuroprotective 5α-pathway (allopregnanolone) homeostasis remained preserved, contrasting with peripheral metabolic saturation. Preferential hippocampal accumulation of 3-oxo and 3β-isomers suggests autonomous regulatory buffering via oxidative 17β-hydroxysteroid dehydrogenase (HSD17B) enzymes, protecting against excessive GABAergic inhibition. This unique pharmacokinetic profile—combining metabolic segregation with active central buffering—defines PG as a dual-mechanism delivery system that generates central neuroactive metabolites—several with previously established GABAergic and neuroprotective activity—without disrupting endogenous neurosteroidogenesis, positioning it as a promising neurotherapeutic candidate minimizing physiological steroid homeostasis disruption. Importantly, the present study characterizes the pharmacokinetic and metabolic fate of PG; the neuroprotective efficacy of PG was demonstrated in our prior functional studies using the same model.

Pregnanolone glutamate (PG) is a synthetic neurosteroid analog showing promise for treating ischemic brain injury, yet its blood–brain barrier (BBB) transport and metabolic fate remain unclear. We investigated the pharmacokinetics of PG in postnatal day 12 rats of both sexes subjected to ET‑1–induced focal hippocampal ischemia. Animals received PG (1 mg/kg i.p.) or vehicle; serum and hippocampal steroidomes were profiled 60 minutes post-administration using GC-MS/MS (hippocampus: n=16 PG+, n=27 PG-; multi-tissue subset: n=6 PG+, n=21 PG-). Our data reveal a "dual-fate" mechanism: PG undergoes rapid systemic hydrolysis as a prodrug but also crosses the BBB intact, with significant parent conjugate accumulation in hippocampus (42.3 pmol/g). The brain functioned as a "metabolic sink," passively accumulating peripherally generated metabolites—such as 17-hydroxypregnanolone—despite local absence of synthesizing enzymes (e.g., CYP17A1). Crucially, PG induced "metabolic segregation" within the CNS: the pharmacological 5β-pathway was saturated (~170-fold pregnanolone increase), while endogenous neuroprotective 5α-pathway (allopregnanolone) homeostasis remained preserved, contrasting with peripheral metabolic saturation. Preferential hippocampal accumulation of 3-oxo and 3β-isomers suggests autonomous regulatory buffering via oxidative HSD17B enzymes, protecting against excessive GABAergic inhibition. This unique pharmacokinetic profile—combining metabolic segregation with active central buffering—defines PG as a dual-mechanism delivery system that generates central neuroactive metabolites without disrupting endogenous neurosteroidogenesis, positioning it as a promising neurotherapeutic candidate minimizing physiological steroid homeostasis disruption.

Focused ultrasound stimulation (FUS) is a promising non-invasive neuromodulation technique that can influence neuronal activity through mechanical stimulation. In this study, primary cortical neurons were isolated from embryonic rat brains and cultured for 14 days in vitro before being divided into Control, FUS 5 V, and FUS 10 V groups. Cells were exposed to low-intensity pulsed FUS (300 kHz, 10 min) using a vertically mounted transducer positioned 5 mm above the culture dish. Post-exposure analyses included cell viability using the MTS assay, total protein quantification by the Bradford method, morphological assessment by Trypan Blue staining, and Fluo-3 AM–based confocal calcium imaging. FUS treatment produced no significant differences in viability or total protein concentration compared with the Control group. Morphological observations confirmed healthy neuronal somata and intact neuritic networks across all groups, with no evidence of cell death or structural damage compared with controls. In contrast, calcium imaging revealed a robust transient elevation in intracellular Ca²⁺ responsiveness when assessed 24 h after FUS exposure, with a significantly higher integrated area under the curve relative to Control. These findings demonstrate that low-intensity FUS safely enhances intracellular calcium signalling while preserving neuronal viability, protein integrity, and morphology, defining a safe acoustic window for non-destructive neuromodulation and providing a framework for mechanistic studies in neurodegenerative disease models.

Background: Postictal refractoriness, i.e., the inability to elicit a new epileptic seizure immediately after the first one, is present in mature animals. Immature rats did not exhibit this refractoriness, and it is replaced by postictal potentiation. In addition to the immediate postictal potentiation, there is a delayed potentiation present at both ages. These phenomena were studied using cortical epileptic afterdischarges as a model. Objective: We aimed to analyze participation of adenosine A1 receptors in postictal potentiation and depression. Methods: Adenosine A1 receptors were studied by means of Western blotting in the cerebral cortex with a focus on the age groups studied electrophysiologically. Stimulation and recording electrodes were implanted epidurally in 12- and 25-day-old rats. The first stimulation always induced conditioning epileptic afterdischarge (AD), and 1 min after its end, the stimulation was repeated to elicit the second, testing AD. Then, the drugs were administered and paired stimulations were repeated 10 min later. A selective agonist CCPA (0.5 and 1 mg/kg i.p.) and a selective antagonist DPCPX (0.1, 0.5 and 1 mg/kg i.p.) were used to examine the possible participation of adenosine A1 receptors. Results: Control younger animals exhibited potentiation of the testing AD and a moderate increase in both conditioning and testing ADs after an injection of saline. The A1 receptor agonist CCPA shortened both post-drug ADs, and neither potentiation was present. The administration of an antagonist DPCPX resulted in marked prolongation of the conditioning AD (delayed potentiation), and the second testing AD was shorter than the post-drug conditioning AD, i.e., there was no longer immediate potentiation of ADs. To eliminate effects of the solvent dimethylsulfoxide, we added experiments with DPCPX suspended with the help of Tween 80. The results were similar, only the prolongation of ADs was not as large, and the testing ADs were significantly depressed. The older control group exhibited a nearly complete suppression of the first testing AD. There was no significant change in the conditioning and testing ADs after CCPA (delayed potentiation was blocked). Both groups of DPCPX-treated rats (with DMSO or Tween) exhibited significant augmentation of delayed potentiation but no significant difference in the immediate depression. Adenosine A1 receptors were present in the cerebral cortex of both age groups, and their quantity was higher in 12- than in 25-day-old animals. Conclusions: An agonist of the A1 receptor CCPA suppressed both types of postictal potentiation in 12-day-old rats, whereas the A1 antagonist DPCPX suppressed immediate potentiation but markedly augmented the delayed one. Immediate postictal refractoriness in 25-day-old rats was only moderately (non-significantly) affected; meanwhile, the delayed potentiation was strongly augmented.

LiCl/pilocarpine status epilepticus (SE) induced in immature rats leads, after a latent period, to hippocampal hyperexcitability. The excitability may be influenced by adenosine, which exhibits anticonvulsant activity. The concentration of adenosine is regulated by adenosine kinase (ADK) present in two isoforms—ADK-L and ADK-S. The main goal of the study is to elucidate the changes in ADK isoform expression after LiCl/pilocarpine SE and whether potential changes, as well as inhibition of ADK by 5-iodotubercidin (5-ITU), may contribute to changes in hippocampal excitability during brain development. LiCl/pilocarpine SE was elicited in 12-day-old rats. Hippocampal excitability in immature rats was studied by the model of hippocampal afterdischarges (ADs), in which we demonstrated the potential inhibitory effect of 5-ITU. ADs demonstrated significantly decreased hippocampal excitability 3 days after SE induction, whereas significant hyperexcitability after 20 days compared to controls was shown. 5-ITU administration showed its inhibitory effect on the ADs in 32-day-old SE rats compared to SE rats without 5-ITU. Moreover, both ADK isoforms were examined in the immature rat hippocampus. The ADK-L isoform demonstrated significantly decreased expression in 12-day-old SE rats compared to the appropriate naïve rats, whereas increased ADK-S isoform expression was revealed. A decreasing ADK-L/-S ratio showed the declining dominance of ADK-L isoform during early brain development. LiCl/pilocarpine SE increased the excitability of the hippocampus 20 days after SE induction. The ADK inhibitor 5-ITU exhibited anticonvulsant activity at the same age. Age-related differences in hippocampal excitability after SE might correspond to the development of ADK isoform levels in the hippocampus.

Pregnanolone glutamate (PA-G) is a neuroactive steroid that has been previously demonstrated to be a potent neuroprotective compound in several biological models in vivo. Our in vitro experiments identified PA-G as an inhibitor of N-methyl-D-aspartate receptors and a potentiator of γ-aminobutyric acid receptors (GABA_ARs). In this study, we addressed the hypothesis that combined GABA_AR potentiation and NMDAR antagonism could afford a potent anticonvulsant effect. Our results demonstrated the strong age-related anticonvulsive effect of PA-G in a model of pentylenetetrazol-induced seizures. PA-G significantly decreased seizure severity in 12-day-old animals, but only after the highest dose in 25-day-old animals. Interestingly, the anticonvulsant effect of PA-G differed both qualitatively and quantitatively from that of zuranolone, an investigational neurosteroid acting as a potent positive allosteric modulator of GABA_ARs. Next, we identified 17-hydroxy-pregnanolone (17-OH-PA) as a major metabolite of PA-G in 12-day-old animals. Finally, the administration of PA-G demonstrated direct modulation of unexpected neurosteroid levels, namely pregnenolone and dehydroepiandrosterone sulfate. These results suggest that compound PA-G might be a pro-drug of 17-OH-PA, a neurosteroid with a promising neuroprotective effect with an unknown mechanism of action that may represent an attractive target for studying perinatal neural diseases.

A bulk of data suggest that the gut microbiota plays a role in a broad range of diseases, including those affecting the central nervous system. Recently, significant differences in the intestinal microbiota of patients with epilepsy, compared to healthy volunteers, have been reported in an observational study. However, an active role of the intestinal microbiota in the pathogenesis of epilepsy, through the so‐called “gut–brain axis,” has yet to be demonstrated. In this study, we evaluated the direct impact of microbiota transplanted from epileptic animals to healthy recipient animals, to clarify whether the microbiota from animals with epilepsy can affect the excitability of the recipients’ brain by lowering seizure thresholds. Our results provide the first evidence that mice who received microbiota from epileptic animals are more prone to develop status epilepticus, compared to recipients of “healthy” microbiota, after a subclinical dose of pilocarpine, indicating a higher susceptibility to seizures. The lower thresholds for seizure activity found in this study support the hypothesis that the microbiota, through the gut–brain axis, is able to affect neuronal excitability in the brain.

The GluN2A subunit of N-methyl-D-aspartate (NMDA) receptors becomes dominant during postnatal development, overgrowing the originally dominant GluN2B subunit. The aim of our study was to show changes of anticonvulsant action of the GluN2A subunit-preferring antagonist during postnatal development of rats. Possible anticonvulsant action of GluN2A-preferring antagonist of NMDA receptors P = [[[(1S)-1-(4-bromophenyl)ethyl]amino](1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl]phosphonic acid tetrasodium salt (PEAQX) (5, 10, 20 mg/kg s.c.) was tested in 12-, 18-, and 25-day-old rats in three models of convulsive seizures. Pentylenetetrazol-induced generalized seizures with a loss of righting reflexes generated in the brainstem were suppressed in all three age groups in a dose-dependent manner. Minimal clonic seizures with preserved righting ability exhibited only moderately prolonged latency after the highest dose of PEAQX. Anticonvulsant action of all three doses of PEAQX against cortical epileptic afterdischarges (generated in the forebrain) was found in the 25-day-old animals. The highest dose (20 mg/kg) was efficient also in the two younger groups, which might be due to lower specificity of PEAQX and its partial affinity to the GluN2B subunit. Our results are in agreement with the postero-anterior maturation gradient of subunit composition of NMDA receptors (i.e., an increase of GluN2A representation). In spite of the lower selectivity of PEAQX, our data demonstrate, for the first time, developmental differences in comparison with an antagonist of NMDA receptors with a dominant GluN2B subunit.