The new antiepileptic drugs lamotrigine and felbamate are effective in phenytoin-resistant kindled rats

We evaluated the anticonvulsant efficacy of the antiepileptic drugs (AEDs) lamotrigine (LTG) and felbamate (FBM) in amygdala kindled rats that had been preselected with respect to their response to phenytoin. Anticonvulsant response was tested by determining the afterdischarge threshold (ADT), i.e., a sensitive measure for drug effects on focal seizure activity. By repeated testing with the phenytoin prodrug fosphenytoin, 3 groups of kindled rats were separated: rats in which consistent anticonvulsant effects were obtained (phenytoin responders), rats which showed no anticonvulsant response (phenytoin nonresponders), and rats with variable responses (variable phenytoin responders). The latter, largest group was used to evaluate at which doses LTG and FBM exerted significant anticonvulsant effects on ADT 1 h after i.p. drug administration. Effective doses were then used for drug testing in phenytoin responders and nonresponders. Both LTG and FBM proved to be effective anticonvulsant drugs in the kindling model by markedly increasing the ADT. Seizure severity and duration recorded at ADT currents were hardly reduced, indicating that both drugs predominantly affect induction of focal seizures and not seizure spread from the focus. In phenytoin nonresponders, LTG and FBM significantly increased ADT, which is in line with their proven efficacy in patients with refractory partial epilepsy in whom phenytoin has failed. However, LTG and, more markedly, FBM were clearly more efficacious in increasing ADT in phenytoin responders than in nonresponders, substantiating that the difference in phenytoin response between these groups of kindled rats extends to other AEDs. The data in this study reveal that phenytoin nonresponders are a unique model for the search for new AEDs with improved efficacy in refractory partial epilepsy.     

A pharmacological study in the kindling model of epilepsy

The anticonvulsant properties of carbamazepine were evaluated in the kindled amygdaloid seizure model in rats. Carbamazepine significantly raised the threshold for seizures, reduced the duration of elicited afterdischarges and attenuated the severity of seizures in previously-kindled rats, at doses that did not cause sedation or ataxia. A similar reduction in the duration of elicited afterdischarges and severity of seizures was seen after suprathreshold stimulation (400 mu A) with doses of carbamazepine that were without obvious sedative or ataxic effects. After acute intraperitoneal injections (solvent = 2% Tween-80 and 70% propylene glycol), the maximum anticonvulsant effectiveness against suprathreshold stimulation was seen at 30 min. When administered daily (13 days) during acquisition or development of kindling, carbamazepine (25 and 50 mg/kg, i.p.) had variable effects on kindling. Neither dose consistently reduced the duration of elicited afterdischarges during the acquisition phase. Both groups tended to reduce the developing seizure, with the smaller dose of carbamazepine (25 mg/kg) resulting in a more consistent and significant reduction in severity of seizures. No significant differences in number of daily stimulations needed to reach fully kindled seizures were found. Previous studies have reported variable results with carbamazepine and the kindled amygdaloid seizure in rats. The present study provides a comprehensive evaluation of carbamazepine in this model of epilepsy and discusses the results with regard to the finding reported previously.     

Morphological alterations of neurons and astrocytes and changes in emotional behavior in pentylenetetrazol-kindled rats.

Changes of emotional behavior and neuronal cell loss in the hippocampus were investigated after pentylenetetrazol (PTZ) induced kindling in rats. Behavioral and morphological changes were studied in partially and fully kindled rats and after different postkindling periods comparing to the controls. The resident-intruder test indicated a diminished offensive behavior in partially and fully kindled animals. The open-field and the cat-odor exposition tests reveal changes in defensive behavioral pattern only in fully kindled rats. A decrease of exploratory locomotion and an increase in freezing were assessed in the open-field and the cat-odor exposition test, respectively, up to 10 weeks after the end of kindling. The first damaged neurons (CA4 region) were observed in the partially kindled group (PK), correlating with an increase in the glial fibrillary acidic protein (GFAP)-immunoreactivity (GFAP-IR) and hypertrophy of astrocytes. The most significant increase in the number of damaged neurons was detected 24 h after completion of kindling (selective vulnerability: CA4/CA1>DG>CA2+CA3). The neuronal loss went on for 10 weeks postkindling. A low correlation between the number of Stage 4 kindling seizures and the number of damaged hippocampal neurons was found 24 h after the end of kindling in individual rats. The present results demonstrate that PTZ kindling goes along with long-lasting changes in emotional behavior. The alterations of the defensive behavior after the termination of kindling can be interpreted as depression-like and are obviously associated with a characteristic pattern of neuronal loss in various hippocampal regions.     

Phenytoin’s effect on the spread of seizure activity in the amygdala kindling model

Phenytoin is a major antiepileptic drug for treatment of limbic seizures. The effect of phenytoin on the generation and spread of seizure activity was studied in a rat model of this type of seizures. Sprague-Dawley and Wistar rats were implanted with a stimulation and recording electrode in the basolateral amygdala. Naive Sprague-Dawley rats showed an increase in current intensity necessary for eliciting afterdischarges (afterdischarge threshold) of about 200% after administration of phenytoin (75 mg/kg i.p.), while seizure severity at threshold was increased compared to controls. Afterdischarge and seizure durations were significantly prolonged under phenytoin. This result suggests that phenytoin can exert a potent anticonvulsant effect on the generation of focal seizure activity, but it does not suppress or may even increase on-going afterdischarge activity once it occurs. Following amygdala kindling in Wistar rats, administration of phenytoin again resulted in an increase in the afterdischarge threshold. However, all rats still showed generalized seizures, and epileptic afterdischarges could be recorded in various limbic brain regions at threshold current. This result suggests that phenytoin can increase the threshold for generation of epileptic discharges in kindled rats, but is not able to prevent the development of generalized seizure activity and the spread of afterdischarges within the limbic system when focal activity is initiated. We conclude that phenytoin is able to suppress focal seizure activity in the amygdala kindling model of the rat. However, it does not prevent the spread of seizure activity originating in the limbic system. Therefore, a decrease in focal seizure susceptibility seems to be the primary target for phenytoin’s anticonvulsant action. Received: 7 March 1997 / Accepted: 17 June 1997     

Kinetic changes and modulation by carbamazepine on voltage-gated sodium channels in rat CA1 neurons after epilepsy

AIM: To study whether the functional properties of sodium channels, and subsequently the channel modulation by carbamazepine (CBZ) in hippocampal CA1 neurons can be changed after epileptic seizures. METHODS: We used the acutely dissociated hippocampal CA1 pyramidal cells from epilepsy model rats 3 weeks and 3 months respectively after kainate injection, and whole-cell voltage-clamp techniques. RESULTS: After long-term epileptic seizures, both sodium channel voltage-dependence of activation and steady-state inactivation shifted to more hyperpolarizing potentials, which resulted in the enlarged window current; the membrane density of sodium current decreased and the time constant of recovery from inactivation increased. CBZ displayed unchanged efficacy on sodium channels, with a similar binding rate to them, except that at higher concentrations, the voltage shift of inactivation was reduced. For the short-term kainate model rats, no differences were detected between the control and epilepsy groups. CONCLUSION: These results indicate that the properties of sodium channels in acutely dissociated hippocampal neurons could be changed following long-term epilepsy, but the alternation might not be enough to induce the channel resistance to CBZ.     

Histidine enhances carbamazepine action against seizures and improves spatial memory deficits induced by chronic transauricular kindling in rats

AIM: To investigate whether histidine can enhance the anticonvulsant efficacy of carbamazepine (CBZ) and simultaneously improve the spatial memory impairment induced by transauricular kindled seizures in Sprague-Dawley rats. METHODS: Chronic transauricular kindling was induced by repeated application of initially subconvulsive electrical stimulation through ear-clip electrodes once every 24 h until the occurrence of 3 consecutive clonic-tonic seizures. An 8-arm radial maze (4 arms baited) was used to measure spatial memory, and histamine and gamma-amino-butyric acid levels were measured by high performance liquid chromatography (HPLC). RESULTS: Chronic transauricular kindling produced a significant impairment of spatial memory and a marked decrease in histamine content in the hypothalamus, the brainstem, and the hippocampus. Injection of histidine (1000 mg/kg or 1500 mg/kg, ip) significantly inhibited transauricular kindled seizures. Injection of histidine at lower doses (200 mg/kg or 500 mg/kg, ip) had no appreciable anticonvulsant effect when administered alone, whereas it significantly potentiated the protective effects of CBZ against kindled seizures. CBZ had no ameliorative effect on memory deficit, but, in contrast, histidine (200 mg/kg or 500 mg/kg, ip) alone or co-administered with CBZ significantly ameliorated the memory deficits induced by the seizures. CONCLUSION: Chronic transauricular kindling is a very useful animal model for evaluating memory deficits associated with epilepsy, and histidine has both a potentiate effect on the anticonvulsant efficacy of CBZ and an ameliorative effect on the spatial memory deficits induced in this model. Histidine at a specific dosage range might serve as a beneficial adjuvant for the clinical treatment of epilepsy, especially when accompanied by impaired spatial memory.     

Multidrug resistance-associated protein 1 decreases the concentrations of antiepileptic drugs in cortical extracellular fluid in amygdale kindling rats

Aim:

To investigate whether multidrug resistance-associated protein 1 (MRP1) was responsible for drug resistence in refractory epilepsy in amygdale kindling rats.

Methods:

Rat amygdale kindling was used as a model of refractory epilepsy. The expression of MRP1 mRNA and protein in the brains was examined using RT-PCR and Western blot. MRP1-positive cells in the cortex and hippocampus were studied with immunohistochemical staining. The rats were intraperitoneally injected with phenytoin (50 mg/kg) or carbamazepine (20 mg/kg), and their concentrations in the cortical extracellular fluid were measured using microdialysis and HPLC. Probenecid, a MRP1 inhibitor (40 mmol/L, 50 μL) was administered through an inflow tube into the cortex 30 min before injection of the antiepileptic drugs.

Results:

The expression of MRP1 mRNA and protein was significantly up-regulated in the cortex and hippocampus in amygdale kindling rats compared with the control group. Furthermore, the number of MRP1-positive cells in the cortex and hippocampus was also significantly increased in amygdale kindling rats. Microdialysis studies showed that the concentrations of phenytoin and carbamazepine in the cortical extracellular fluid were significantly decreased in amygdale kindling rats. Pre-administration of probenecid could restore the concentrations back to their control levels.

Conclusion:

Up-regulation of MRP1 is responsible for the resistance of brain cells to antiepileptic drugs in the amygdale kindling rats.     

New aspects in the pharmacology of kindling implications for mechanism of of action in kindling

Kindling is a long-lasting, transsynaptic, pathway-specific plastic change in brain function. It has been proposed as a model of neural plasticity, learning and memory, as well as a model of epilepsy. To elucidate the action of substances characterized by their ability to improve learning and memory and to have an activatory, protective and function restoring effect on nerve cells in distress, the effects of nootropic drugs and of antioxidative acting substances on the development of kindling and the seizure behavior in the kindled state were investigated. Nootropic drugs suppress the development of pentetrazol (PTZ)- and amygdala-kindling and possess anticonvulsive potency preferentially in kindled rats. In comparison to amygdala-kindling the substances were found to be more effective against PTZ-kindled seizures. Chemically different antioxidants in doses known to scavenge free radicals suppress markedly the development of kindling and possess anticonvulsant potency in chemically and electrically kindled rats. The results provide arguments in favor of functional alterations of transmission relevant membrane processes with structural and functional reorganization of membrane constituents and lead to the assumption that the changes mainly in part rest on a selective and focal involvement of free radicals. It is proposed that enhanced calcium entry into neurons and consequent biochemical alterations connected with a focal increase in free radicals is part of the mechanisms underlying kindling phenomenon.     

Pharmacological modification of amygdaloid-kindled seizures

The acute effect of several antiepileptic drugs on amygdaloid-kindled seizures was investigated in rats. Phenobarbital, diazepam and trimethadione produced a dose-dependent decrease in severity and amygdaloid afterdischarge duration (ADD) of full kindled seizures. In contrast, phenytoin did not suppress kindled seizures but appeared to increase seizure severity and ADD, suggesting that its action is fundamentally different from that of the other antiepileptic agents. The general anesthetic, ketamine, was weakly effective in abolishing established kindled seizures but had a marked ability to prevent kindling when given prophylactically from the outset of amygdaloid stimulation. The anti-kindling action of ketamine may be related to its ability to enhance central noradrenergic mechanisms.     

癫清片对戊四唑点燃大鼠海马损害的病理学观察

目的观察癫清片对戊四唑(PTZ)点燃大鼠脑组织学损害特点。方法取按照One’s分级标准造模成功的癫大鼠60只,随机分为模型对照组;癫清片高、中、低剂量组;癫宁片组及苯妥英钠片组,连续灌胃给药28 d,处死后脑标本制成4μm层厚的横轴位切片,HE染色,光镜下观察各组海马的CA1、CA2、CA3、CA4的组织学改变,并选择组织学损害最明显的区域在高倍镜下(40×10)计数神经元的丢失。结果模型对照组总的神经细胞数量减少,变性神经细胞可占锥体细胞的83%,各给药组与模型组相比,均可增加总的神经细胞数量,变性神经细胞比率减少,其中癫清片高剂量组与苯妥英钠片组表现相似。结论PTZ可使海马的CA1、CA2、CA3、CA4的大量神经细胞变性坏死;癫清片具有增加锥体层总体神经细胞和正常神经细胞数量的作用。