Elsevier

Epilepsy & Behavior

Volume 48, July 2015, Pages 4-14
Epilepsy & Behavior

Effect of GLT-1 modulator and P2X7 antagonists alone and in combination in the kindling model of epilepsy in rats

https://doi.org/10.1016/j.yebeh.2015.04.056Get rights and content

Highlights

  • Effect of GLT-1 modulator and P2X7 antagonist against PTZ-kindling was explored.

  • Functional crosstalk between P2X7 receptors and GLT-1 was elucidated.

  • New therapeutic strategy for epilepsy without neurotoxic and cognitive side effects

Abstract

Introduction

Multiple lines of investigation have explored the role of glutamatergic and purinergic systems in epilepsy, related cognitive impairment, and oxidative stress. Glutamate transporters, particularly GLT-1 expression, were found to be decreased, and purinergic receptor, P2X7 expression, was found to be increased in brain tissue associated with epilepsy. The present study was carried out to investigate the effect of ceftriaxone (GLT-1 upregulator) and Brilliant Blue G (P2X7 antagonist) against PTZ-induced kindling in rats. The study was further extended to elucidate the cross-link between glutamatergic and purinergic pathways in epilepsy.

Material and methods

Systemic administration of subconvulsant dose of PTZ (30 mg/kg) every other day for 27 days (14 injections) significantly increased the mean kindling, and developed generalized tonic–clonic seizures, and reduced motor co-ordination, cognitive skills, oxidative defense (increases lipid peroxidation, nitrite levels and decreases GSH level) and acetylcholinesterase enzyme activities in the cortex and subcortical region. Treatments with CEF (100 and 200 mg/kg) and BBG (15 and 30 mg/kg) alone and in combination (CEF 100 mg/kg and BBG 15 mg/kg) significantly decreased the mean kindling score and restored behavioral and oxidative defense activities compared with treatment with PTZ.

Conclusions

The combination of both the drugs was shown to have better effect in preventing kindled seizures and a significantly synergistic effect compared with their effect alone in PTZ-kindled rats. The present study elucidated the mechanistic role of GLT-1 modulator and selective P2X7 antagonist and their combination against PTZ-induced kindling. The study for the first time demonstrated the cross-link between glutamatergic and purinergic pathways in epilepsy treatment.

Introduction

Epilepsy a common neurological disorder characterized by episodes of neurologic dysfunction in which abnormal neuronal firing is manifest clinically by changes in motor control, sensory perception, behavior, and/or autonomic function that apparently result from complex processes involving several neurotransmitters [1], [2]. Changes occurring during epileptogenesis contribute to network imbalances between excitation and inhibition which result in the enduring predisposition to the generation of epileptic seizures [3]. Excitation is modulated by excitatory neurotransmitters and mostly by glutamate [4]. It is generally accepted that the majority of glutamate transport in the CNS, particularly as related to excitatory transmission, is mediated by a group of high-affinity, sodium-dependent, glutamate transporters, thereby terminating the transmitter signal and protecting neurons from an excitotoxic action of glutamate and providing cells throughout the body with glutamate for metabolic purposes [5], [6]. Approximately 80% of the glutamate transporters expressed in the hippocampus are GLT-1 [7]. Although expressed primarily by astrocytes, GLT-1 is also expressed on neuronal axon terminals. The expression of GLT-1a in axon terminals has potentially important implications for the physiology of excitatory synaptic transmission in regulating synaptic glutamate, maintaining glutamate stores in the presynaptic terminal, interacting with glutamate receptors, contributing a glutamate-regulated anionic conductance to the plasma membrane of the presynaptic bouton, and controlling crosstalk between excitatory synapses. GLT-1b is present to a lesser extent [8]. Relatively little is known about the mechanisms that regulate GLT-1 or the other Na+-dependent glutamate transporters, but it suggests that the expression of GLT-1/EAAT-2 is regulated by transcriptional and/or posttranscriptional processes [6]. It was suggested that malfunctioning of glutamate transporters (GLTs) is one of the main causative factors of hyperexcitation, seizure spread, and neurotoxicity [6], [9], [10], [11]. Some studies also reported that an increased GLT-1 expression can protect mice against status epilepticus (SE)-induced death, neuropathological changes, and chronic seizure development [12], [13]. On the basis of previous reports, we can speculate that enhancing GLT-1/EAAT2 protein expression is a potential therapeutic approach to treat epilepsy.

The beta-lactam antibiotics, such as ceftriaxone (CEF), enhance the ex vivo expression of a neuroprotective protein GLT-1 in a concentration-dependent manner [14]. This CEF-induced GLT-1 upregulation blocks the metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) at the mossy fiber (MF)–CA3 hippocampal synapse [15].

Brilliant Blue G (Coomassie Brilliant Blue G) (BBG) is a selective noncompetitive antagonist of P2X7 type of purinergic receptors and is well tolerated by human beings [16], [17]. The increased expression of P2X7 R is found in the hippocampus, mainly within mossy fibers and the dentate gyrus of the chronically epileptic rat [18]. There is a crosstalk between purinergic and glutamatergic pathways, as it has been demonstrated that the Na+-influx triggered by the activation of P2X7 affects the activities of glutamate transporters via a reduction in the amplitude of transporter currents. Stimulation of P2X7 signaling is shown to downregulate the activity for glutamate transport through GLT-1 transporters by triggering posttranscriptional regulation of GLAST/GLT-1 expression via phosphoinositol 3-kinase cascade and production of oxidants [19], [20].

Kindling is a model of epilepsy produced by repeated administration of an initially subconvulsive electrical or chemical stimulus that results in an increase in seizure activity, culminating in a generalized seizure [21]. The electrographic and behavioral components of kindled seizures are thought to be similar to human partial-onset seizures, as the focal component of the kindled seizure can progress into a generalized seizure. In this model, the effect of drugs on both focal and generalized seizure types can be assessed.

With this background, the present study was conducted to explore the effect of CEF (GLT-1 modulator) and BBG (P2X7 antagonist) administration on seizure development induced by repetitive administration of subconvulsant dose (30 mg/kg) of PTZ in rats. The effect of each drug alone and in combination on epilepsy-induced cognitive impairment and oxidative stress in PTZ-kindling model in rats was also assessed.

Section snippets

Experimental animals

Wistar rats, weighing 200–250 g, were used in the present study. Animals were obtained from the Central Animal House facility of ISF College of Pharmacy, Moga, and housed in a group of three in polypropylene cages with husk bedding under standard conditions of light and dark cycle with food and water ad libitum. Animals were acclimatized to laboratory conditions before behavior observations. All the behavioral assessments were carried out between 9:00 h and 15:00 h. The experimental protocol was

Protective effect of CEF, BBG, and their combination on PTZ-induced mean kindling score in rats

Repeated measures two-way ANOVA analysis indicated an overall significant effect of treatment (F6,40 = 66.92; P < 0.001) and time (F13,40 = 67.03; P < 0.001) (Fig. 1). Chronic, subconvulsive PTZ (30 mg/kg) administration on alternate days for a total period of 27 days (14 injections) produced kindling in rats. The maximum score was reached on day 27 of the study when all the animals in the PTZ control group showed the 5th stage two consecutive times and developed generalized tonic–clonic seizures.

Discussion

In the present study, the PTZ-kindling model was used to explore behavior, biochemical, and cellular alterations that are involved in the pathogenesis of epilepsy. The study was further extended to compare the potential role of the GLT-1 modulator (CEF) and that of a P2X7 receptor antagonist (BBG) against PTZ-induced kindling cognitive impairment, motor impairment, and oxidative stress. Chemical kindling induced by PTZ produces a rodent model of generalized epilepsy [32]. Microdialysate

Conclusion

We conclude that the administration of CEF and BBG either individually or in combination prevents PTZ-kindled seizures in rats and associated cognitive impairment and restores oxidative defense and acetylcholinesterase activity. We also found that the combination-treated group and other high-dose-treated groups and, in some cases, low-dose-treated groups are demonstrated better or improved learning, memory, and motor activity in rats compared with the standard drug diazepam. This suggests that

Acknowledgment

We would like to express our thanks to Mr. Parveen Garg, Chairman, I.S.F. College of Pharmacy for providing us with excellent research facilities.

Conflict of interest

The authors have no conflicts of interest to disclose.

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