Received Date: 17/02/2014 Accepted Date: 07/04/2014 Article Type: Original Article Adjuvant Anticholinesterase Therapy for the Management of Epilepsy-inducedMemory Deficit:A Critical Preclinical Study Awanish Mishra and Rajesh Kumar Goel Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Punjab, India (Received 17 February 2014; Accepted 7 May 2014)
Author for correspondence: Rajesh KumarGoel,Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala-147002, Punjab, India (fax: 0175-2283073, e-mail: [email protected]). Abstract: Epilepsy is one of the major neurological disorders still awaiting safer drugs with improved antiepileptic effect and lesser side effects. Apart from epilepsy itself, AEDs also have been shown to induce cognitive impairment in patients with epilepsy. There are limited data for the treatment of this menace. As cholinergic approach has widely been practiced for the restoration of memory in various neurodegenerative disorders, hence this study was envisaged to evaluate add on effect of acetylcholinesterase inhibitor (tacrine) with phenytoin in pentylenetetrazole-kindling induced learning and memory deficit in mice. In this study, mice were kindled using subconvulsive dose of pentylenetetrazole (35 mg/kg, i.p.; at interval of 48±2 hr) andsuccessfully kindled animals were divided into different groups and treated with vehicle, phenytoinand phenytoinin combination with tacrine (0.3 mg/kg), atropine (1 mg/kg) and tacrine + atropine. Effect of different interventions on learning and memory was evaluated using elevated plus maze and passive shock avoidance on days 5, 10, 15 and 20. Phenytoin-treated kindled animals were associated with learning and memory deficit, while tacrinesupplementation improved memory deficit with increased seizure severity score. Atropine treatment significantly reversed the protective effect of tacrine. Neurochemical findingsalso support the behavioural finding of the study. Our results suggest the use of
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/bcpt.12275 This article is protected by copyright. All rights reserved.
anticholinesterases, with better seizuretolerance, for the management of cognitive impairment of epilepsy, as adjunct therapy.
1. Epilepsy is one of the most debilitating, non-communicable, chronic neurological disorders, with enormous variations in aetiology and clinical features, resulting in asymmetrical episodic bursts of electrical activity in certain neurons spreading to the entire brain. Chronic epileptic patients are more often coupled with one or more psychiatric co-morbidities. Out of these psychiatric co-morbidities, learning and memory deficit come forward as one of most debilitating neurobehavioural co-morbidity of epilepsy [1-2]. Learning and memory deficit, affects around 30% of epileptic patients [3], greatly contributes to the major adverse effect on overall health, quality of life and substantially increase health care costs in these patients [4]. Albeit, there is wide recognition of epidemiological aspects of cognitive comorbidities of epilepsy but clinical interventions to prevent co-morbidities for the management of epileptic patients are issues of substantial concern. Current management of epilepsy appears to be ineffective with available antiepileptic drugs in terms of their effectiveness to control their associated cognitive side effects [5-7]. Under normal conditions, cholinergic afferents in hippocampus and cortex, essential brain parts involved in initiation of seizures and development of learning and memory, play a pivotal role in the control of neuronal excitability [8], learning and memory processes [9]. Gradual loss of hippocampal cholinergic neurotransmission causing progressive deterioration of memory results in Alzheimer’s disease [10] and similar depletion in hippocampal acetylcholine level has been implicated in an experimental model of epilepsy [11]. Preliminary psychoneurochemical investigations in our laboratory suggested that depleted hippocampal acetylcholine level in pentylenetetrazole-kindled animals might be due to enhanced acetylcholinesterase activity [12]. Enhanced cholinergic innervations have been suggested to improve memory by choline supplementation in status epilepticus[13]. The above findings suggest that an anticholinesterase approach may be useful for management of epilepsy-associated memory deficit. Anticholinesterase treatment per se has major limitation to provoke seizures [14],and therefore it was presumed that combination treatment of anticholinesterase with standard antiepileptic drug may be more useful. Tacrine, an anticholinesterase, has been reported to reduce memory complications associated with pentylenetetrazole-kindling and facilitated kindling procedure when This article is protected by copyright. All rights reserved.
administered along with pentylenetetrazole[15]. This supports the use of tacrine for the management of learning and memory deficit in post pentylenetetrazole-kindling (experimental model of epilepsy providing opportunity to study progressive cognitive changes with a close resemblance to clinical epilepsy)[12,16-18] as adjuvant therapy.Thus, in continuation of our previous study to find a comprehensive or add on target for treatment of epilepsy associated memory deficit [12], this study was envisaged to explore the effect of supplementation of tacrine with phenytoin in pentylenetetrazole-kindling-induced learning and memory deficit as adjuvant approach.
2. Methods 2.1 Drugs and chemicals Pentylenetetrazole, Tacrine was procured from Sigma-Aldrich, Co., St. Louis, MO, USA. Phenytoin sodium was received as a gift sample from Jackson Laboratories Ltd., India and atropine was received from Q. P. Pharmachem, Derabassi, India. 2.2 Animals The studies were carried out on male Swiss Albino mice (22-28g) and were obtained from the breeder (ChaudharyCharan Singh Haryana Agricultural University, Hisar, Haryana, India). Swiss Albino mice were housed in standard cages at room temperature (22±2°C), under natural light/dark cycle and with free access to water and food (standard laboratory pellets) before the experiments. The experimental protocol was duly approved by the Institutional Animal Ethics Committee (IAEC) and the care of the animals was carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India vide protocol approval no. 107/99/CPCSEA/-2009-4.2. 2.3 Induction of Kindling Induction of kindling was done by the method previously validated in our laboratory [12,16]. Briefly, pentylenetetrazole (dissolved in warm saline, i.p.) was injected in the pentylenetetrazole-kindled group at a sub-convulsive dose of 35 mg/kg at 48±2 hr intervalfor 9-11 weeks or until the animal showedappearance of tonic-clonic convulsion after two consecutive pentylenetetrazole administrations. 2.4 Experimental Protocol A total of 60 animals were employed in this study. Group I, naïve animals, consisted of untreated animals (n=10) and the remainder50 animals were subjected to pentylenetetrazolekindling. Excluding resistant and mortality, only successfully kindled animals were further This article is protected by copyright. All rights reserved.
randomly divided into 5 groups. Group II (vehicle control group) consisted of kindled animals receiving normal saline (10 ml/kg/day; i.p.; n=8).Group III (phenytoin per se group) consisted of kindled animals treated with phenytoin (30 mg/kg/day; i.p.;n=7).Group IV consisted of kindled animals receiving phenytoin (30 mg/kg/day) and tacrine (0.3 mg/kg/day; i.p.; n=8).Group V consisted of kindled animals receiving phenytoin and atropine (10 mg/kg/day; i.p.; n=7).Group VI consisted of kindled animals receiving phenytoin, tacrine and atropine (n=8). The treatment schedule was followed up to 20 days. All the kindled animals (Group II, III, IV, V and VI) were challenged with subconvulsive dose of Pentylenetetrazole (35 mg/kg; i.p.) on day 5, 10, 15 and 20 and corresponding seizure severity score was recorded using the modified Racine’s scale [16,17]. After 2 hr of pentylenetetrazole challenging dose, once their locomotor activity became normalized, the animals were evaluated for their performance in elevated plus maze and passive shock avoidance paradigm on days 5, 10, 15 and 20. After behavioural assessments on day 20, all the animals were killed after 4hr of the last pentylenetetrazole injection for neurochemical analysis. 2.5 Behavioural Assessments 2.5.1 Transfer latency in Elevated Plus Maze Spatial memory was evaluated by recording transfer latency using elevated plus maze on days 0, 10, 15 and 20 following the procedure previously standardized in our laboratory[12,16]. 2.5.2 Number of Mistakes and Step-Down Latency in Passive Shock Avoidance Paradigm For the evaluation of contextual fear memory, modified passive shock avoidance paradigm previously standardized in our laboratory was used [12,16,17]. The retrieval of learned task was evaluated by recording the changes in the number of mistakes and step-down latency on day 5, 10, 15 and 20. 2.6 Neurochemical Estimations After behavioural evaluation on day 20 (4hr after the last pentylenetetrazole injection), all animals were killed by cervical dislocation and their brains were dissected to isolate cortex and hippocampus. Isolated brain parts were weighed and subdivided into two equal portions, and homogenates were prepared for the estimation of amino acids (glutamate and GABA), monoamines (noradrenaline, dopamine and serotonin) using the HPLC-FD method, total nitrite level and acetylcholinesterase activity using the microplate reader method previously standardized in our laboratory [12]. This article is protected by copyright. All rights reserved.
2.7 Statistical Analysis The statistical analysis was performed using the Sigma Stat Statistical Software ,version 3.5. Statistical significance was calculated using one-way ANOVA followed by the StudentNewman-Keulstest. Each value was expressed as mean ± S.E.M. and statistical significance was considered at P< 0.05. 3.
Results
3.1 Effect on Seizure Severity Score The significant change in seizure severity score was observed on day 5 (F(5,42) = 84.484; P < 0.001), day 10 (F(5,42) = 28.643; P < 0.001), day 15 (F(5,42) = 36.773; P < 0.001) and day 20 (F(5,42) = 44.724; P < 0.001) in different groups. On days 5, 10, 15 and 20, after administration of pentylenetetrazole,the challenging doses in vehicle-treated animals showed significant increase (P
Author for correspondence: Rajesh KumarGoel,Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala-147002, Punjab, India (fax: 0175-2283073, e-mail: [email protected]). Abstract: Epilepsy is one of the major neurological disorders still awaiting safer drugs with improved antiepileptic effect and lesser side effects. Apart from epilepsy itself, AEDs also have been shown to induce cognitive impairment in patients with epilepsy. There are limited data for the treatment of this menace. As cholinergic approach has widely been practiced for the restoration of memory in various neurodegenerative disorders, hence this study was envisaged to evaluate add on effect of acetylcholinesterase inhibitor (tacrine) with phenytoin in pentylenetetrazole-kindling induced learning and memory deficit in mice. In this study, mice were kindled using subconvulsive dose of pentylenetetrazole (35 mg/kg, i.p.; at interval of 48±2 hr) andsuccessfully kindled animals were divided into different groups and treated with vehicle, phenytoinand phenytoinin combination with tacrine (0.3 mg/kg), atropine (1 mg/kg) and tacrine + atropine. Effect of different interventions on learning and memory was evaluated using elevated plus maze and passive shock avoidance on days 5, 10, 15 and 20. Phenytoin-treated kindled animals were associated with learning and memory deficit, while tacrinesupplementation improved memory deficit with increased seizure severity score. Atropine treatment significantly reversed the protective effect of tacrine. Neurochemical findingsalso support the behavioural finding of the study. Our results suggest the use of
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/bcpt.12275 This article is protected by copyright. All rights reserved.
anticholinesterases, with better seizuretolerance, for the management of cognitive impairment of epilepsy, as adjunct therapy.
1. Epilepsy is one of the most debilitating, non-communicable, chronic neurological disorders, with enormous variations in aetiology and clinical features, resulting in asymmetrical episodic bursts of electrical activity in certain neurons spreading to the entire brain. Chronic epileptic patients are more often coupled with one or more psychiatric co-morbidities. Out of these psychiatric co-morbidities, learning and memory deficit come forward as one of most debilitating neurobehavioural co-morbidity of epilepsy [1-2]. Learning and memory deficit, affects around 30% of epileptic patients [3], greatly contributes to the major adverse effect on overall health, quality of life and substantially increase health care costs in these patients [4]. Albeit, there is wide recognition of epidemiological aspects of cognitive comorbidities of epilepsy but clinical interventions to prevent co-morbidities for the management of epileptic patients are issues of substantial concern. Current management of epilepsy appears to be ineffective with available antiepileptic drugs in terms of their effectiveness to control their associated cognitive side effects [5-7]. Under normal conditions, cholinergic afferents in hippocampus and cortex, essential brain parts involved in initiation of seizures and development of learning and memory, play a pivotal role in the control of neuronal excitability [8], learning and memory processes [9]. Gradual loss of hippocampal cholinergic neurotransmission causing progressive deterioration of memory results in Alzheimer’s disease [10] and similar depletion in hippocampal acetylcholine level has been implicated in an experimental model of epilepsy [11]. Preliminary psychoneurochemical investigations in our laboratory suggested that depleted hippocampal acetylcholine level in pentylenetetrazole-kindled animals might be due to enhanced acetylcholinesterase activity [12]. Enhanced cholinergic innervations have been suggested to improve memory by choline supplementation in status epilepticus[13]. The above findings suggest that an anticholinesterase approach may be useful for management of epilepsy-associated memory deficit. Anticholinesterase treatment per se has major limitation to provoke seizures [14],and therefore it was presumed that combination treatment of anticholinesterase with standard antiepileptic drug may be more useful. Tacrine, an anticholinesterase, has been reported to reduce memory complications associated with pentylenetetrazole-kindling and facilitated kindling procedure when This article is protected by copyright. All rights reserved.
administered along with pentylenetetrazole[15]. This supports the use of tacrine for the management of learning and memory deficit in post pentylenetetrazole-kindling (experimental model of epilepsy providing opportunity to study progressive cognitive changes with a close resemblance to clinical epilepsy)[12,16-18] as adjuvant therapy.Thus, in continuation of our previous study to find a comprehensive or add on target for treatment of epilepsy associated memory deficit [12], this study was envisaged to explore the effect of supplementation of tacrine with phenytoin in pentylenetetrazole-kindling-induced learning and memory deficit as adjuvant approach.
2. Methods 2.1 Drugs and chemicals Pentylenetetrazole, Tacrine was procured from Sigma-Aldrich, Co., St. Louis, MO, USA. Phenytoin sodium was received as a gift sample from Jackson Laboratories Ltd., India and atropine was received from Q. P. Pharmachem, Derabassi, India. 2.2 Animals The studies were carried out on male Swiss Albino mice (22-28g) and were obtained from the breeder (ChaudharyCharan Singh Haryana Agricultural University, Hisar, Haryana, India). Swiss Albino mice were housed in standard cages at room temperature (22±2°C), under natural light/dark cycle and with free access to water and food (standard laboratory pellets) before the experiments. The experimental protocol was duly approved by the Institutional Animal Ethics Committee (IAEC) and the care of the animals was carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India vide protocol approval no. 107/99/CPCSEA/-2009-4.2. 2.3 Induction of Kindling Induction of kindling was done by the method previously validated in our laboratory [12,16]. Briefly, pentylenetetrazole (dissolved in warm saline, i.p.) was injected in the pentylenetetrazole-kindled group at a sub-convulsive dose of 35 mg/kg at 48±2 hr intervalfor 9-11 weeks or until the animal showedappearance of tonic-clonic convulsion after two consecutive pentylenetetrazole administrations. 2.4 Experimental Protocol A total of 60 animals were employed in this study. Group I, naïve animals, consisted of untreated animals (n=10) and the remainder50 animals were subjected to pentylenetetrazolekindling. Excluding resistant and mortality, only successfully kindled animals were further This article is protected by copyright. All rights reserved.
randomly divided into 5 groups. Group II (vehicle control group) consisted of kindled animals receiving normal saline (10 ml/kg/day; i.p.; n=8).Group III (phenytoin per se group) consisted of kindled animals treated with phenytoin (30 mg/kg/day; i.p.;n=7).Group IV consisted of kindled animals receiving phenytoin (30 mg/kg/day) and tacrine (0.3 mg/kg/day; i.p.; n=8).Group V consisted of kindled animals receiving phenytoin and atropine (10 mg/kg/day; i.p.; n=7).Group VI consisted of kindled animals receiving phenytoin, tacrine and atropine (n=8). The treatment schedule was followed up to 20 days. All the kindled animals (Group II, III, IV, V and VI) were challenged with subconvulsive dose of Pentylenetetrazole (35 mg/kg; i.p.) on day 5, 10, 15 and 20 and corresponding seizure severity score was recorded using the modified Racine’s scale [16,17]. After 2 hr of pentylenetetrazole challenging dose, once their locomotor activity became normalized, the animals were evaluated for their performance in elevated plus maze and passive shock avoidance paradigm on days 5, 10, 15 and 20. After behavioural assessments on day 20, all the animals were killed after 4hr of the last pentylenetetrazole injection for neurochemical analysis. 2.5 Behavioural Assessments 2.5.1 Transfer latency in Elevated Plus Maze Spatial memory was evaluated by recording transfer latency using elevated plus maze on days 0, 10, 15 and 20 following the procedure previously standardized in our laboratory[12,16]. 2.5.2 Number of Mistakes and Step-Down Latency in Passive Shock Avoidance Paradigm For the evaluation of contextual fear memory, modified passive shock avoidance paradigm previously standardized in our laboratory was used [12,16,17]. The retrieval of learned task was evaluated by recording the changes in the number of mistakes and step-down latency on day 5, 10, 15 and 20. 2.6 Neurochemical Estimations After behavioural evaluation on day 20 (4hr after the last pentylenetetrazole injection), all animals were killed by cervical dislocation and their brains were dissected to isolate cortex and hippocampus. Isolated brain parts were weighed and subdivided into two equal portions, and homogenates were prepared for the estimation of amino acids (glutamate and GABA), monoamines (noradrenaline, dopamine and serotonin) using the HPLC-FD method, total nitrite level and acetylcholinesterase activity using the microplate reader method previously standardized in our laboratory [12]. This article is protected by copyright. All rights reserved.
2.7 Statistical Analysis The statistical analysis was performed using the Sigma Stat Statistical Software ,version 3.5. Statistical significance was calculated using one-way ANOVA followed by the StudentNewman-Keulstest. Each value was expressed as mean ± S.E.M. and statistical significance was considered at P< 0.05. 3.
Results
3.1 Effect on Seizure Severity Score The significant change in seizure severity score was observed on day 5 (F(5,42) = 84.484; P < 0.001), day 10 (F(5,42) = 28.643; P < 0.001), day 15 (F(5,42) = 36.773; P < 0.001) and day 20 (F(5,42) = 44.724; P < 0.001) in different groups. On days 5, 10, 15 and 20, after administration of pentylenetetrazole,the challenging doses in vehicle-treated animals showed significant increase (P
