Purslane protects against the reproductive toxicity of carbamazepine treatment in pilocarpine-induced epilepsy model

Objective: To investigate the protective effect of purslane with carbamazepine treatment.Methods: Male albino rats were modulated by pilocarpine to be epileptic.Both the normal and epileptic rats were treated with carbamazepine, purslane or carbamazepine plus purslane, with separate non-treated control groups for both normal and epileptic rats.Results: The data from the current study showed amelioration in amino acids and electrolytes in the epileptic rats treated with purslane and carbamazepine, with this amelioration occurring without decreasing the fertility hormones(testosterone,dehydroepiandrosterone, luteinizing hormone and follicle stimulating hormone).Purslane treatments also prevented the increase in estradiol.The decreased epileptic hyperexcitability with purslane was evidenced by decreased glial fibrillary acidic protein and lipid peroxidation.Conclusions: Natural products like purslane could be used with the highly repetitive drugs like carbamazepine to reduce or prevent its side-effects.

Correlation Study on Expression of GST-π Protein in Brain Tissue and Peripheral Blood of Epilepsy Rats Induced by Pilocarpine

Previous studies have suggested that glutathione-S-transferase π （GST-π） over-expression in the brain tissue is associated with refractory epilepsy. However, whether the change in GST-π level in the peripheral blood is in line with that in brain tissue remains unknown. This study examined the correlation between GST-π in brain tissue and that in peripheral blood in rat models of pilocarpine-induced refractory epilepsy. The animals were divided into drug-resistant group and drug-responsive group according to the response to anti-epileptic drugs. GST-π expression in brain tissue was immunohistochemically determined, while the expression of GST-π in peripheral blood was analyzed by Western blotting. In the hippocampus and cortex, GST-π was mainly found in the cytoplasm and membrane of neurons, and the GST-π expression level was higher in drug-resistant group than in the drug-responsive group and saline control group （P〈0.05）. Moreover, there was no significant difference between responders and saline control animals （P〉0.05）. The change in expression of GST-π in peripheral blood showed the same pattern as that in brain tissues, suggesting GST-π might contribute to drug resistance in epilepsy. Importantly, the GST-π over-expression in peripheral blood could be used as a marker for resistance to anti-epileptic agents.

Proliferation changes in hippocampal neurons following pilocarpine-induced intractable epilepsy in adult rats

Epilepsy can lead to the changes in neurons residing in the dentate gyrus. The present study aimed to observe the cell dividing features following epilepsy in adult rat hippocampi, and to study difference in cell proliferation between adult rats with common epilepsy and intractable epilepsy.Adult, male, Sprague Dawley rats ware randomly divided into control （n = 8, treatment with normal saline） and three expenmental groups： common epilepsy （n = 33）, intractable epilepsy （n = 11）, and drug-responsive （n = 25）. Pilocarpine （15 mg/kg） was intrapentoneally administered to establish epilepsy in the three experimental groups. Rats that developed epilepsy were treated with chloral hydrate. Rats that did not exhibit spontaneous seizures were enrolled in the common epilepsy group, and rats with spontaneous seizure were included in the spontaneous seizure group. At 6 hours after epileptic attack termination, rats ware intraperitoneally injected with bromodeoxyuridine （BrdU; 50 mg/kg）, an optimal marker forlabeling cell proliferation in vivo, four times.Immunohistochemistry results at 48 hours after BrdU injection indicated that the number of BrdU-positive cells was the highest in the common epilepsy group, followed by the control group,and lastly the intractable group （P 〈 0.01）. In addition, the number of BrdU-positive cells in the common epilepsy group was similar to the drug-responsive group. The present findings demonstrated that intractable epilepsy led to decreased hippocampal neurons in adult rats when compared to common epilepsy.

Cytotoxicity of pilocarpine to human corneal stromal cells and its underlying cytotoxic mechanisms

AIM： To examine the cytotoxic effect of pilocarpine, an anti-glaucoma drug, on human corneal stromal（HCS）cells and its underlying cytotoxic mechanisms using an in vitro model of non-transfected HCS cells.· METHODS： After HCS cells were treated with pilocarpine at a concentration from 0.15625 g/L to 20.0 g/L,their morphology and viability were detected by light microscopy and MTT assay. The membrane permeability,DNA fragmentation and ultrastructure were examined by acridine orange（AO）/ethidium bromide（EB） double-staining. DNA electrophoresis and transmission electron microscopy（TEM）, cell cycle, phosphatidylserine（PS）orientation and mitochondrial transmembrane potential（MTP） were assayed by flow cytometry（FCM）. And the activation of caspases was checked by ELISA.· RESULTS： Morphology observations and viability assay showed that pilocarpine at concentrations above0.625 g/L induced dose- and time-dependent morphological abnormality and viability decline of HCS cells. AO/EB double-staining, DNA electrophoresis and TEM noted that pilocarpine at concentrations above 0.625 g/L induced dose- and/or time-dependent membrane permeability elevation, DNA fragmentation, and apoptotic body formation of the cells. Moreover, FCM and ELISA assays revealed that 2.5 g/L pilocarpine also induced S phase arrest, PS externalization, MTP disruption, and caspase-8,-9 and-3 activation of the cells.· CONCLUSION： Pilocarpine at concentrations above0.625 g/L（1/32 of its clinical therapeutic dosage） has a dose- and time-dependent cytotoxicity to HCS cells by inducing apoptosis in these cells, which is most probably regulated by a death receptor-mediated mitochondrion-dependent signaling pathway.

A Single Hypoxic Event Ameliorates Pilocarpine Induced Hyperkinetic Movements in Planaria

Intermittent hypoxia or hypoxia therapy is exposing an individual to oxygenation conditions that are below atmospheric levels in a planned or acute timeframe. Hypoxia therapy is a potentially novel therapeutic strategy for a variety of pathologies including: mitochondrial disorders, exercise training, and mild cognitive impairments. Mitochondrial dysfunction, hyperkinetic movements, and cognitive impairments are hallmarks of seizures and status epilepticus (SE). A seizure can be considered uncontrolled electrical activity in the brain and SE is a seizure lasting more than 30 minutes, or multiple seizures without regaining consciousness in between. We examined the possibility of using the Pilocarpine model for seizure like activity on brown planaria (Dugesia tigrine). Pilocarpine is a muscarinic acetylcholine receptor agonist capable of creating seizure related brain damage. We utilized 5 mM dosages of pilocarpine and then measured open field behaviour for 3 minutes. Mobility and aversive hyperkinetic movements were observed throughout the measurement phase. After exposure to 5 mM pilocarpine, the planaria displayed behaviours consistent with seizures (e.g. aversive hyperkinetic movements and decreased mobility). Additionally, we measured the effects of an acute hypoxic event on Planaria behaviour. We used 25% carbonated water to create a hypoxic environment for the planaria and then measured mobility and hyperkinetic movements for 3 minutes. We noted that exposure to the hypoxic en-vironment produced no changes in behaviour. However, the aversive hyperkinetic move-ments produced with pilocarpine administration were completely absent when a brief (3 minutes) hypoxic episode followed the pilocarpine exposure (p < 0.05). Aversive behav-iours remained present when the ordering of pilocarpine and hypoxia were counterbal-anced. This ordering effect was consistent across 40 trials. Further evaluation of the pilo-carpine seizure model and intermittent hypoxia on planarian behaviour is warranted.

Anticonvulsive and antioxidant effects of curcumin on pilocarpine-induced seizures in rats

Background Curcumin, an active ingredient of turmeric with antioxidant and anti-inflammatory properties has recently been reported to have anticonvulsant effects in several animal models of epilepsy. This study aimed to investigate the effects of curcumin on the pilocarpine rat model of status epilepticus. Methods The effect of intraperitoneal administration of curcumin （30, 100, and 300 mg/kg） on pilocarpine-induced seizures in rats was tested. The correlation between seizure activity and hippocampal levels of nitric oxide synthase and free radicals was quantified. Whether curcumin treatment modulated these parameters was also investigated. Results Curcumin significantly increased seizure threshold at doses of 100 and 300 mg/kg. Rats with pilocarpine- induced seizures showed significantly elevated levels of malonaldehyde, nitric oxide synthase, and lactate dehydrogenase, but decreased levels of superoxide dismutase and glutathione compared with normal control rats. At doses of 100 and 300 mg/kg, curcumin reversed the effects of pilocarpine-induced seizures on nitric oxide synthase, lactate dehydrogenase, glutathione, and superoxide dismutase. However, curcumin did not restore the elevated malonaldehyde levels. Conclusion Curcumin has anticonvulsant activity in the pilocarpine rat model of seizures, and that modulation of free radicals and nitric oxide synthase may be involved in this effect.

One hour of pilocarpine-induced status epilepticus is sufficient to develop chronic epilepsy in mice, and is associated with mossy fiber sprouting but not neuronal death

Determining the minimal duration of status epilepticus （SE） that leads to the development of subsequent spontaneous seizures （i.e., epilepsy） is important, because it provides a critical timewindow for seizure intervention and epilepsy prevention. In the present study, male ICR （imprinting Control Region） mice were injected with pilocarpine to induce acute sei zures. SE was terminated by diazepam at 10 min, 30 min, 1 h, 2 h and 4 h after seizure onset. Spon taneous seizures occurred in the 1, 2 and 4 h SE groups, and the seizure frequency increased with the prolongation of SE. Similarly, the Morris water maze revealed that the escape latency was significantly increased and the number of target quadrant cross ings was markedly decreased in the 1, 2 and 4 h SE groups. Robust mossy fiber sprouting was observed in these groups, but not in the 10 or 30 min group. In contrast, FluoroJade B staining revealed significant cell death only in the 4 h SE group. The incidence and frequency of spontaneous seizures were corre lated with Timm score （P = 0.004） and escape latency （P = 0.004）. These data suggest that SE longer than one hour results in spontaneous motor seizures and memory deficits, and spontaneous seizures are likely associated with robust mossy fiber sprouting but not neuronal death.

Serotonin 1A receptor inhibits the status epilepticus induced by lithium-pilocarpine in rats

Status epilepticus （SE） is a life-threatening neurological emergency associated with a high mortality rate. The serotonin 1A （5-HTIA） receptor is a possible target for the treatment of SE, but its role in animal models and the precise area of brain involved remain controversial. The hippocampus is a candidate site due to its key role in the development of SE and the existence of a high density of 5-HT1A receptors. Therefore, we investigated the effects of subcutaneous and intrahippocampal activation of 5-HT1A receptors in lithium-pilocarpine-induced SE, and tested whether the hippocampus is a true effector site. We developed SE in male Sprague-Dawley rats by giving lithium chloride （LiCI; 3 meq/kg, i.p.） 22-24 h prior to pilocarpine （25 mg/kg, i.p.）, and found that 8-OH-DPAT, a 5-HT1A receptor agonist administered subcutaneously （s.c.） at 0.5 or 1.0 mg/kg 1 h before pilocarpine injection increased the latency to the first epileptiform spikes, the electrographic SE, and the behavioral generalized seizures （GS）, while reducing the total EEG seizure time （P 〈0.01）. The duration of GS was shortened only by 1.0 mg/kg 8-OH-DPAT s.c. （P 〈0.05）. All these effects were inhibited by combined administration of WAY-100635 （1.0 mg/kg, s.c.） （P 〈0.05）, an antagonist of the 5-HT1A receptor, but WAY-100635 alone and low doses of 8-OH- DPAT （0.01 and 0.1 mg/kg） did not alter seizure activity. Furthermore, intrahippocampal 8-OH-DPAT only shortened the GS duration （P 〈0.05）. These findings imply that the 5-HT1A receptor is a promising therapeutic target against the generation and propagation of SE, and hippocampal receptors are involved in reducing the seizure severity.

Sphingosine 1-phosphate receptor 1 regulates bloodbrain barrier permeability in epileptic mice

Destruction of the blood-brain barrier is a critical component of epilepsy pathology.Several studies have demonstrated that sphingosine 1-phosphate receptor 1 contributes to the modulation of vascular integrity.However,its effect on blood-brain barrier permeability in epileptic mice remains unclear.In this study,we prepared pilocarpine-induced status epilepticus models and pentylenetetrazol-induced epilepsy models in C57BL/6 mice.S1P1 expression was increased in the hippocampus after status epilepticus,whereas tight junction protein expression was decreased in epileptic mice compared with controls.Intraperitoneal injection of SEW2871,a specific agonist of sphingosine-1-phosphate receptor 1,decreased the level of tight junction protein in the hippocampus of epileptic mice,increased blood-brain barrier leakage,and aggravated the severity of seizures compared with the control.W146,a specific antagonist of sphingosine-1-phosphate receptor 1,increased the level of tight junction protein,attenuated blood-brain barrier disruption,and reduced seizure severity compared with the control.Furthermore,sphingosine 1-phosphate receptor 1 promoted the generation of interleukin-1βand tumor necrosis factor-αand caused astrocytosis.Disruption of tight junction protein and blood-brain barrier integrity by sphingosine 1-phosphate receptor 1 was reversed by minocycline,a neuroinflammation inhibitor.Behavioral tests revealed that sphingosine 1-phosphate receptor 1 exacerbated epilepsy-associated depression-like behaviors.Additionally,specific knockdown of astrocytic S1P1 inhibited neuroinflammatory responses and attenuated blood-brain barrier leakage,seizure severity,and epilepsy-associated depression-like behaviors.Taken together,our results suggest that astrocytic sphingosine 1-phosphate receptor 1 exacerbates blood-brain barrier disruption in the epileptic brain by promoting neuroinflammation.

几种经典化学致癫痫实验动物模型概述

本文回顾了KA、PTZ和Pilocarpine三种经典化学致癫痫的实验动物模型的建立及特征，并对其实际应用和特点进行了简单比较与讨论。

Thermal influence of saliva secretion <i>ex vivo</i>in the mouse submandibular gland

The physiological and pharmacological responses of an ex vivo mouse submandibular gland were used to study fluid secretion and cell signaling in response to muscarinic stimulation at increasing temperatures. Saliva production at 37°C was 5.5-fold that at 25°C with pilocarpine stimulation and 9.8-fold that at 25°C with cevimeline stimulation. Both of these muscarinic agonists are used clinically. With the experimental agonist carbachol (CCh), saliva secretion was increased with an increase in temperature, but the CCh concentration producing the peak flow was the same in both dose-response curves, suggesting that the muscarinic receptor itself is not responsible for the temperature dependence. Purinergic agonists also induced temperature-dependent saliva production ex vivo. The calcium ionophore A23187 failed to have a significant effect on saliva production. The CCh-induced increase in intracellular Ca2+ also upregulated the initial increase and sustained the plateau phase of saliva flow. Thus, muscarinic receptor stimulation of saliva production is temperature sensitive due to an increase in intracellular Ca2+.