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Abstract: The present study assessed the effect of carbamazepine and lamotrigine on cognitive function and oxidative stress in brain during chemically induced epileptogenesis in rats. Epileptogenesis was induced by administration of pentylenetetrazole (30 mg/kg, s.c.) on alternate days (three times/week) for 9–11 weeks or until stage 4 of seizure score was achieved. The neurobehavioural parameters used for cognitive assessment were step-down latency in continuous avoidance apparatus and transfer latency in elevated plus maze test paradigm. Carbamazepine and lamotrigine were administered intraperitoneally in doses of 60 mg/kg and 25 mg/kg, respectively, according to the groups, once a day for 11 weeks. Oxidative stress was assessed in isolated homogenized whole brain samples and estimated for the levels of malondialdehyde, reduced glutathione, catalase and superoxide dismutase. The results showed that lamotrigine did not produce any change in cognitive function, while carbamazepine produced cognitive dysfunction. Cognitive decline seen in the carbamazepine-treated pentylenetetrazole-kindled group was also associated with increased oxidative stress. Lamotrigine treatment had no effect on oxidative stress parameters alone, while it significantly decreased oxidative stress in the pentylenetetrazole-kindled group as compared to the pentylenetetrazole-kindled carbamazepine-treated group.
Epilepsy is a disorder of the central nervous system (CNS) and affects nearly 1% of the world population. Epilepsy is characterized by paroxysmal cerebral dysrhythmia manifesting as brief episodes of loss or disturbance of consciousness with or without characteristic convulsive body movements, at times accompanied by sensory or psychiatric phenomenon. These episodes are called seizures and it is suggested that such episodes, when appearing in a cluster of CNS neurons, will sometimes signal abnormally. The seizures may be due to abnormalities in the brain during development of central neurons leading to an imbalance of nerve signaling chemicals called neurotransmitters which may be excitatory (glutamate) or inhibitory (GABA), disruption of cell membrane surrounding neurons or brain-derived neurotrophic factor mediated activation of tyrosine kinase B in the hippocampus [1–3].
Currently available treatments cure 80% of the patients by drugs and surgery and 20% have intractable seizures. The current treatment for epilepsy includes psychotherapy, pharmacotherapy and surgery, out of which pharmacotherapy remains the mainstay. Antiepileptic drugs, being used in therapy, result in numerous side effects in the patient, e.g. rashes, megaloblastic anaemia, sedation, dizziness, ataxia, etc. A considerable number of antiepileptic drugs induce cognitive and behavioural abnormalities like impairment of learning and memory. Also, cognitive abnormalities occur more often in patients with intractable seizures and usually appear on long-term administration of antiepileptic drugs. Long-term use of certain antiepileptic drugs has also been shown to increase oxidative stress . For example, valproic acid has been shown to increase lipid peroxidation in patients with epilepsy . On the other hand, some antiepileptic drugs have been shown to cause decrease in oxidative stress; e.g. diphenylhydantoin has been shown to cause increase in glutathione reductase activity and decrease in oxidative stress in patients with epilepsy .
On the background of these observations, the present study was conducted to investigate the effect of carbamazepine and lamotrigine on cognitive function and oxidative stress in rats and to determine if oxidative stress is involved in causing cognitive decline. Oxidative stress was assessed by measuring four parameters, viz malondialdehyde, reduced glutathione and antioxidative enzymes superoxide dismutase and catalase in the rat brain.
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It has been reported that epilepsy as well as the drugs which are used to treat this CNS disorder can produce several adverse outcome, among which the cognitive decline and increased oxidative stress are particularly important factors that significantly affect the patient’s quality of life . Cognitive decline has long been recognized as a sequel of intractable epilepsy [18,19]. However, it is not clear whether it is the phenomenon of epileptogenesis or oxidative stress or both which might be contributing to the decline in cognitive function. Some antiepileptic drugs used in the treatment have been reported to improve cognitive functioning in patients with epilepsy by themselves or by providing good seizure control as a result of their antiepileptogenic action . However, recent studies have shown consistently that antiepileptic treatment may have adverse cognitive consequences [21,22]. Moreover, the cognitive side effects represent the long-term outcome of antiepileptic drug therapy. The present study was conducted to compare the effects of carbamazepine with lamotrigine, the two commonly used antiepileptic drugs on cognitive function by measuring step-down latency in continuous avoidance test paradigm and transfer latency on elevated plus-maze apparatus, the two experimental paradigms which are commonly used models for the assessment of memory function in animals . The experimental epileptogenesis was induced by chronic administration of pentylenetetrazole in rats .
The results showed that the step-down latency was significantly decreased and transfer latency was significantly increased in pentylenetetrazole-kindled rats as compared to the control group of animals which suggested significant memory impairment in the pentylenetetrazole-kindled rats. This observation is in line with reported clinical studies that showed significant cognitive decline in epileptic patients . When carbamazepine was administered to the pentylenetetrazole-kindled and non-kindled groups of rats, both groups showed significant decrease in step-down latency and increase in transfer latency, thereby suggesting a cognitive impairment in both groups of animals. There was no significant difference in step-down latency and transfer latency values between the carbamazepine-treated pentylenetetrazole-kindled and carbamazepine-treated non-kindled groups suggesting that carbamazepine was unable to prevent cognitive decline produced during pentylenetetrazole epileptogenesis. It appears that carbamazepine itself caused cognitive dysfunction in non-kindled group of rats. In some previous studies, treatment with carbamazepine was observed to derange the cognitive function in both epileptic and normal healthy volunteers [25,26].
When lamotrigine was administered to the pentylenetetrazole-kindled and non-kindled groups of rats, the pentylenetetrazole-kindled lamotrigine-treated group showed significant decrease in step-down latency and increase in transfer latency as compared to the control group. The lamotrigine-treated non-kindled group showed no significant change in step-down latency and transfer latency as compared to the normal control group. Such an outcome of lamotrigine effect suggested that the memory impairment in the lamotrigine-treated kindled group was due to a process of epileptogenesis and not due to the lamotrigine treatment. When the two antiepileptic drugs, lamotrigine and carbamazepine treatments in the non-kindled groups of rats were compared with each other, a significant difference in step-down latency and transfer latency was observed, i.e. the carbamazepine-treated non-kindled group showed a significant decrease in step-down latency and increase in transfer latency as compared to the lamotrigine-treated non-kindled group. This comparison suggests a cognitive dysfunction produced by carbamazepine alone vis-a-vis lamotrigine. These results may be attributed to change in anxiety-related behaviour also. However, as the results obtained in both transfer latency and step-down latency parameters are similar, it is more likely that the results are due to changes in cognition. When comparing the effect of these two drugs, it was clearly demonstrated that lamotrigine itself caused no impairment of memory or cognitive decline. The above observations further confirmed the results of some previous studies which had shown no neuropsychological adverse effect of lamotrigine on cognitive function when used in epileptic patients .
The process of epileptogenesis and long-term use of certain antiepileptic drugs has been shown in previous studies to cause increase in reactive oxygen species leading to oxidative stress and neuronal damage in patients with epilepsy . On the background of these observations, the effect of carbamazepine and lamotrigine on oxidative stress in rat brain was investigated. This was assessed by measuring four parameters of oxidative stress viz malondialdehyde, glutathione, superoxide dismutase and catalase activity in the rat brain.
The malondialdehyde assay is often considered as an index of free radical generation which increases in conditions of oxidative stress . The results showed that the malondialdehyde levels were significantly raised in the pentylenetetrazole-kindled rats as compared to the normal control group. These results confirm the observations of previous studies which had shown increased lipid peroxidation in pentylenetetrazole-induced kindling in rats . The carbamazepine-treated pentylenetetrazole-kindled group also showed significant increase in malondialdehyde levels as compared to the control group. The carbamazepine-treated non-kindled group showed no significant change in malondialdehyde levels as compared to the control group. The lamotrigine-treated pentylenetetrazole-kindled group also showed increase in malondialdehyde levels in rat brain as compared to the control group but it was significantly lower with the values observed in the pentylenetetrazole-kindled carbamazepine-treated group of animals. This could be explained by the additive action of lamotrigine on inhibition of glutamate-mediated excitotoxicity, which would lead to less production of free radicals . A number of previous studies have also shown a considerable oxidative potential of carbamazepine  and antioxidative potential of lamotrigine in rats .
Glutathione is the most prevalent and important intracellular antioxidant. The process of epileptogenesis has been shown to be associated with decreased glutathione concentration in the epileptic focus . On this background, the results showed that glutathione levels were significantly reduced in the pentylenetetrazole-kindled group as compared to the control group. The pentylenetetrazole-kindled rats treated with carbamazepine also showed significant decline in glutathione levels in rat brain as compared to the control group, suggestive of increased oxidative stress. These results are consistent with previous studies which had shown oxidative potential of some first-line antiepileptic drugs such as carbamazepine, phenytoin, etc. which were shown to produce decreased activity of reduced glutathione . The lamotrigine-treated pentylenetetrazole-kindled group also showed significant decrease in glutathione activity as compared to the control group but this decrease was significantly less when compared with the values of the carbamazepine-treated pentylenetetrazole-kindled group of animals. This suggests an antioxidative effect of lamotrigine. The antioxidative effect of lamotrigine could be related to its inhibition of glutamate-mediated excitotoxicity through N-malondialdehyde receptors, as it has been reported to decrease the release of glutamic acid in the face of increased release. The resulted decreased N-malondialdehyde receptor stimulation would then lead to less free radical generation and subsequently a diminished excitotoxicity.
Superoxide dismutase is a major antioxidative enzyme of the brain. The activity of superoxide dismutase has been shown to decline in patients with epilepsy . The results of the present study are in conformity with these observations; accordingly, the activity of superoxide dismutase was observed to decrease significantly in pentylenetetrazole-kindled rats as compared to the control group of animals. The pentylenetetrazole-kindled group treated with carbamazepine also showed significant decline in superoxide dismutase activity as compared to the control group. These results confirm the observations of some of the previous studies which showed an increased potential of first generation antiepileptic drugs (e.g. carbamazepine, phenytoin, etc.) to cause oxidative stress as compared to the second generation antiepileptic drugs (e.g. lamotrigine, zonisamide, etc.) in rat astrocyte cultures in vitro . The pentylenetetrazole-kindled rats treated with lamotrigine also showed a significant decrease in superoxide dismutase enzyme activity as compared to the control group of rats. Further, the lamotrigine-treated non-kindled group showed values of superoxide dismutase enzyme activity comparable to the control group of rats. As compared to the results with reduced glutathione in carbamazepine- and lamotrigine-treated pentylenetetrazole-kindled rats, the effect of lamotrigine was not as marked on superoxide dismutase enzyme activity in the pentylenetetrazole-kindled animals and suggests that lamotrigine has more effect on glutathione than superoxide dismutase activity.
Catalase is an enzyme responsible for detoxification of H2O2 formed by the action of superoxide dismutase. The activity of the enzyme catalase has been shown to decline in epileptic patients . The results of the present study are also in line with these observations and point to a decrease in catalase enzyme activity in pentylenetetrazole-kindled carbamazepine-treated rats. The pentylenetetrazole-kindled rats receiving lamotrigine also showed a decline in catalase enzyme activity than the control group of rats. These results further suggest that lamotrigine has more favourable antioxidative effect via glutathione than via superoxide dismutase or catalase activity.
Thus, the results of this study altogether suggest that lamotrigine does not affect the cognitive function produced by pentylenetetrazole-induced epileptogenesis while carbamazepine caused significant cognitive dysfunction by itself as well as worsens this biochemical assault during epileptogenesis. Also, lamotrigine was shown to have mild antioxidative activity while carbamazepine showed no antioxidant property. It is also evident from the results that oxidative stress may be one of the causative mechanisms of decline in cognitive function during epileptogenesis as well as during antiepileptic drug treatment. Further studies are needed to confirm such conclusions and find out the relationship between epileptogenesis and antiepileptic drug therapy with cognitive dysfunction and this basis of affecting each other.