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.
- Publikační typ
- časopisecké články MeSH
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 (GABAARs). In this study, we addressed the hypothesis that combined GABAAR 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 GABAARs. 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.
- Publikační typ
- časopisecké články MeSH
We have demonstrated previously that activation of either the ETA or ETB receptor can induce acute electrographic seizures following the intrahippocampal infusion of endothelin-1 (ET-1) in immature (P12) rats. We also demonstrated that activation of the ETA receptor is associated with marked focal ischemia, while activation of the ETB receptor is not. Exploring the mechanisms underlying seizures induced by these two ET-1 receptor interactions can potentially provide insight into how focal ischemia in immature animals produces seizures and whether ischemiarelated seizures differ from seizures not associated with ischemia. To explore these seizure mechanisms we used microdialysis to determine biomarkers associated with seizures in P12 rats following the intrahippocampal infusion of two different agents: (1) ET-1, which activates both the ETA and ETB receptors and causes focal ischemia and (2) Ala-ET-1, which selectively activates only the ETB receptor and does not cause ischemia. Our results show that seizures associated with combined ETA and ETB receptor activation (and ischemia) have a different temporal distribution and microdialysis profile from seizures associated with ETB activation alone (and without ischemia). Seizures with combined activation peak within the first hour after infusion and the microdialysis profile is characterized by a significant increase in the ratio of glutamic acid to GABA. By contrast, seizures with activation of only the ETB receptor peak in the second hour after infusion and microdialysis shows a significant increase in the ratio of leukotriene B4 to prostaglandin E2. These findings suggest that ischemia-related seizures in immature animals involve an imbalance of excitation and inhibition, while non-ischemiarelated seizures involve an inflammatory process resulting from an excess of leukotrienes.
- MeSH
- endotelin-1 toxicita MeSH
- hipokampus účinky léků metabolismus MeSH
- ischemie mozku chemicky indukované metabolismus MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- receptor endotelinu A metabolismus MeSH
- receptor endotelinu B metabolismus MeSH
- záchvaty chemicky indukované metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Objective: The adenosinergic system may influence excitability in the brain. Endogenous and exogenous adenosine has anticonvulsant activity presumably by activating A1 receptors. Adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) may thus bolster anticonvulsant effects, but its action and the number of A1 receptors at different developmental stages are not known. Methods: Hippocampal epileptic afterdischarges (ADs) were elicited in 12-, 15-, 18-, 25-, 45-, and 60-day-old rats. Stimulation and recording electrodes were implanted into the dorsal hippocampus. The A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 0.5 or 1 mg/kg) was administered intraperitoneally 10 min before the first stimulation. Control animals were injected with saline. All rats were stimulated with a 2-s series of 1-ms biphasic pulses delivered at 60 Hz with increasing stepwise intensity (0.05-0.6 mA). Each age and dose group contained 9-14 animals. The AD thresholds and durations were evaluated, and the A1 receptors were detected in the hippocampus in 7-, 10-, 12-, 15-, 18-, 21-, 25-, 32-, and 52-day-old rats. Results: Both CCPA doses significantly increased hippocampal AD thresholds in 12-, 15-, 18-, and 60-day-old rats compared to controls. In contrast, the higher dose significantly decreased AD threshold in the 25-day-old rats. The AD durations were significantly shortened in all age groups except for 25-day-old rats where they were significantly prolonged. A1 receptor expression in the hippocampus was highest in 10-day-old rats and subsequently decreased. Significance: The adenosine A1 receptor agonist CCPA exhibited anticonvulsant activity at all developmental stages studied here except for 25-day-old rats. Age-related differences might be due to the development of presynaptic A1 receptors in the hippocampus.
- Publikační typ
- časopisecké články MeSH
The perinatal period of life is a critical moment for a proper development of CNS, however it is a risky period, which is associated with many pathologies. Hence, it is important to acquire knowledge and combine new methodologies for understanding the developmental aspects of physiology/pathophysiology of the brain. We have successfully developed and applied the method of monitoring a tissue metabolism in the immature brain. Using a combination of microdialysis technique and HPLC-MS basal levels of amino acids and other low molecular weight molecules in the hippocampus of immature rats (12-days-old) were mapped. Our results demonstrate the suitability of chosen parameters of sampling and analysis for the monitoring of tissue biochemistry and metabolomic mapping in immature brain. Moreover, a potential of the proposed method can found a place in clinical practice and for biomarkers monitoring of various CNS diseases.
- MeSH
- aminokyseliny MeSH
- mikrodialýza * využití MeSH
- mozek * metabolismus MeSH
- potkani Wistar MeSH
- vysokoúčinná kapalinová chromatografie využití MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Během vývoje mozku dochází v průběhu dospívání k mnoha významným změnám na úrovni molekulární, buněčné i orgánové. Přestože výzkum v této oblasti probíhá již řadu desetiletí, nejsou zcela objasněné některé mechanizmy a probíhající vývojové změny. Mikrodialýza (MD) v kombinaci s vysokoúčinnou kapalinovou chromatografíí a hmotnostní spektrometrií (HPLC/MS) umožňuje monitorování tkáňové biochemie, t.j. metabolomické mapování, v nezralém mozku téměř v reálném čase. Úspěšná aplikace kombinace těchto dvou metod má vysoký a unikátní potenciál rozšířit znalosti v oblasti fyziologie a patofyziologie nezralého mozku a dokonce pomoci vyvinout účinnou léčbu mnoha dětských nemocí.
In early developmental stages of brain, many maturational processes on molecular, cellular and organ level take place. Although many years of research were dedicated to this topic, the clarification of numerous mechanisms and their roles in later development is still lacking. Combination of microdialysis (MD) and high performance liquid chromatography combined with mass spectrometry (HPLC/MS) represents a unique possibility for the monitoring of tissue biochemistry and metabolomic mapping in immature brain in almost real -time condition. Hence, it has a great potential to broaden our knowledge in physiology and pathophysiology of immature brain and can help to develop effective treatment of many infant diseases.
- Klíčová slova
- nezralý mozek,
- MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- metabolomika * MeSH
- mikrodialýza * MeSH
- mozek * metabolismus růst a vývoj MeSH
- novorozenec nedonošený MeSH
- novorozenec MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- práce podpořená grantem MeSH
Mikrodialýza (MD) v kombinaci s vysokoúčinnou kapalinovou chromatografií a hmotnostní spektrometrií (HPLC-MS) je úspěšně využí- vána k metabolomickému mapování dynamických změn v mozku. Potřeba využití těchto metod vzrůstá s rozvojem nových syntetických drog (NSD), kdy je nutné na jejich výskyt velmi rychle reagovat a určit jejich případnou nebezpečnost a jejich adiktivní potenciál. Z uvedeného důvodu by tato metodika mohla v budoucnu sloužit jako prediktivní nástroj pro určení nebezpečnosti NSD na základě metabolických změn v průběhu intoxikace. Dovolujeme si odhadnout, že tato metoda má vysoký potenciál pro rozšíření znalostí nejen o účinku nových syntetických drog (NSD) a molekulární podstatě rozvoje závislosti, ale může také posloužit k vývoji nových léčebných postupů.
In the past decades, the requirement for in vivo detection of diverse chemical substances has increased due to development of new synthetic drugs with an unknown impact on brain metabolism. Microdialysis in combination with high -performance liquid chromatography and mass spectrometry (HPLC-MS) is an efficient tool for monitoring dynamic biochemical changes in the brain. Hence, it could be used for dissecting the mechanism of action of newly appearing synthetic abusive drugs and processes involved in the development of drug addiction. Moreover, it can be optimized for diverse compounds and biological tissues and utilized, for example, for testing of novel therapeutical approaches.
- MeSH
- dopamin sekrece MeSH
- hmotnostní spektrometrie MeSH
- metabolomika MeSH
- methamfetamin farmakologie MeSH
- mikrodialýza * MeSH
- mozek metabolismus MeSH
- poruchy spojené s užíváním psychoaktivních látek * metabolismus MeSH
- potkani Wistar MeSH
- vysokoúčinná kapalinová chromatografie * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
The period around birth is a risky time for stroke in infants, which is associated with two major acute and subacute processes: anatomical damage and seizures. It is unclear as to what extent each of these processes independently contributes to poor outcome. Furthermore, it is unclear whether there is an interaction between the two processes - does seizure activity cause additional brain damage beyond that produced by ischemia and/or does brain damage foster seizures? The model of focal cerebral ischemia induced by the intrahippocampal infusion of endothelin-1 (ET-1) in 12-day-old rat was used to examine the role of the endothelin receptors in the development of focal ischemia, symptomatic acute seizures and neurodegeneration. ET-1 (40pmol/μl) was infused either alone or co-administered with selective antagonists of ETA (BQ123; 70nmol/μl) or ETB receptors (BQ788; 70nmol/1μl). Effects of activation of ETB receptors were studied using selective agonist 4-Ala-ET-1 (40pmol/1μl). Regional cerebral blood flow (rCBF) and tissue oxygenation (pO2) were measured in anesthetized animals with a Doppler-flowmeter and a pO2-sensor, respectively. Seizure development was assessed with video-EEG in freely moving rats. Controls received the corresponding volume of the appropriate vehicle (10mM PBS or 0.01% DMSO-PBS solution; pH7.4). The extent of hippocampal lesion was determined using FluoroJade B staining performed 24h after ET-1 infusion. Infusion of ET-1 or ET-1+ETB receptor antagonist reduced rCBF to ~25% and pO2 to ~10% for about 1.5h, whereas selective ETB agonist, ET-1+ETA antagonist and the PBS vehicle had only negligible effect on the rCBF and pO2 levels. Reduction of rCBF was associated with the development of lesion in the injected hippocampus. In all groups, except sham operated and PBS controls, epileptiform activity was observed after activation of the ETA or the ETB receptors. The data revealed a positive correlation between the severity of morphological damage and all the measured seizure parameters (seizure frequency, average and total seizure duration) in the ET-1 group. In addition, the severity of morphological damage positively correlated with the average seizure duration in animals after infusion of ET-1+ETA receptor antagonist or after infusion of ET-1+ETB receptor antagonist. Our results indicate that the activation of ETA receptors is crucial for ischemia development, however either ETA or ETB receptors mediate the development of seizures following the application of ET-1 in immature rats. The dissociation between the ischemic-producing and seizure-producing processes suggests that damage is not necessary to induce seizures, although it may exacerbate them.
- MeSH
- elektroencefalografie * účinky léků MeSH
- endotelin-1 aplikace a dávkování toxicita MeSH
- hipokampus účinky léků metabolismus MeSH
- injekce intraventrikulární MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- receptor endotelinu A agonisté metabolismus MeSH
- receptor endotelinu B agonisté metabolismus MeSH
- záchvaty chemicky indukované metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
