|Year : 2015 | Volume
| Issue : 1 | Page : 86-89
Protective effect of rutin on impairment of cognitive functions of due to antiepileptic drugs on zebrafish model
Shagun Dubey, Aditya Ganeshpurkar, Divya Bansal, Nazneen Dubey
Shri Ram Institute of Technology-Pharmacy, Near ITI, Madhotal, Jabalpur, Madhya Praesh, India
|Date of Submission||31-Jul-2013|
|Date of Decision||25-Jun-2014|
|Date of Acceptance||19-Dec-2014|
|Date of Web Publication||30-Jan-2015|
Asst. Prof. Aditya Ganeshpurkar
Shri Ram Institute of Technology-Pharmacy, Near ITI, Madhotal, Jabalpur, Madhya Praesh
Source of Support: None, Conflict of Interest: None
Aim: The severity of adverse reactions due to antiepileptics is observed during initiation and early treatment in which impairment of cognitive effects are common. Since long time, herbal medicine is a natural remedy to treat neural symptoms. Phytochemicals have been proven to be potent neuro-protective agents. Rutin, a bioflavonoid is established to be nootropic in many studies. In this study, we aimed to determine the protective effect of rutin in zebrafish against the side effects produced by AEDs.
Materials and Methods: Seizures were induced in zebrafish by phenylenetetrazole. Rutin pretreatment (50 mg/kg, single injection, i.p.) was done before commencement of the study. Behavioral studies were performed as: latency to move high in the tank, locomotion effects, color effect, shoal cohesion, light/dark test on Zebrafish.
Results: Treatment with rutin reverted the locomotor behavior to normal. Treatment with AEDs caused fishes to move in all regions while, in case of treatment with rutin, the response reverted to normal. Treatment with AEDs altered swimming behavior of zebrafish, however, rutin showed a positive effect over this behavior. Treatment with AEDs resulted in restricted movement of zebrafish to the dark zone. Treatment with rutin caused increased latency of zebrafish to move in the light compartment. Similarly, time spent in the light compartment by zebrafish treated with rutin was significantly (P < 0.01) higher as compared to zebrafish treated with AEDs.
Conclusion: The results suggest a protective role of rutin on cognition impaired by AEDs.
Keywords: Cognition, epilepsy, rutin, zebrafish
|How to cite this article:|
Dubey S, Ganeshpurkar A, Bansal D, Dubey N. Protective effect of rutin on impairment of cognitive functions of due to antiepileptic drugs on zebrafish model. Indian J Pharmacol 2015;47:86-9
|How to cite this URL:|
Dubey S, Ganeshpurkar A, Bansal D, Dubey N. Protective effect of rutin on impairment of cognitive functions of due to antiepileptic drugs on zebrafish model. Indian J Pharmacol [serial online] 2015 [cited 2020 Jan 28];47:86-9. Available from: http://www.ijp-online.com/text.asp?2015/47/1/86/150357
| » Introduction|| |
Adverse effects may be mild, acute serious or chronic. The severity of adverse reactions due to antiepileptic drugs (AEDs) is mostly seen during initiation and early treatment. The most common side effects observed with AEDs therapy are CNS adverse effects. These include cognitive effects and psychiatric effects. Cognitive effects typically include diminished memory, attention, intelligence, executive function, language skills, and processing speed. The psychiatric effects include mood disorders, notably anxiety and depression, which are commonly observed.  The most severe side effect is diminished memory, attention, intelligence, executive function, language skills and processing speed. Nootropic agents may be used to treat these disorders related to learning abilities and memory. Thus, there is a need to explore treatments for memory dysfunctions.  Herbal medicine have been used as a natural remedy to treat neural symptoms. Phytochemicals have been proven to be potent neuro-protective agents. In previous studies, researchers found that phytochemicals including sterols, alkaloids, flavonoids, glycosides, saponins, tannins and terpenes are neuroprotective.  Rutin, a well-known flavonoid has been proven to be nootropic in a number of studies. , The present study aimed to determine the neuroprotective effect of rutin with reference to the adverse effects produced by AEDs.
| » Materials and Methods|| |
Potassium chloride, sodium chloride, sodium bicarbonate, sodium phosphate, calcium chloride were purchased from Central Drug House (CDH), India; glucose were purchased from Fischer Scientific (India). Trichloroacetic acid and ethylenediaminetetraacetic acid were purchased from Qualigens, India; thiobarbituric acid was purchased from Sigma, US. All other chemicals used were of analytical grade. Triple distilled water was used in the experiment.
The following drugs were used: Phenytoin (Samarth Pharma, India), Valproic acid (VPA) (Sun Pharma, India), Levetiracetam (Lupin, India). Rutin was purchased from CDH, India.
PTZ model seizures were induced individually in each zebrafish. For this, each fish was exposed to 7.5 mM PTZ in a 250 mL beaker. The seizure-like behavior was assessed according to the stage. These were divided into three stages, stage I dramatic increase in swimming activity, stage II-whirlpool like swimming behavior and stage III clonus like seizures followed by loss of posture i.e complete immobilization for about 1-3 s. , The fish was given PTZ treatment until the commencement of stage III. After stage III has reached, each fish was captured gently from the treatment beaker and were placed in drug beaker used to perform biochemical and molecular analyses. Control group animals were maintained in a 250 mL beaker with tank water for the same period and conditions as the PTZ-treated groups. Before the PTZ exposure, the animals remained exposed to AED treatments for 1 h, enough time for all drugs to achieve seizure suppressor effect. Phenytoin sodium (PHT) (450 μM), VPA (50 mM) and levetiracetam (3 mM) concentrations were chosen based on previous studies. ,
| » Rutin Dosing|| |
Rutin pretreatment (50 mg/kg, single injection, i.p.) was done before commencement of the study. 
The procedures for group behavior task (GBT) were performed in an isolated room. Twenty-Four hours prior to the experiments, both male and female fish (approximately 1:1 distribution) were moved to the experimental room in order to reduce the variance in environment during the behavioral assay. The GBT was performed according to Piato et al.  For the simultaneous evaluation of height in the tank, locomotion, color, and shoal cohesion the test tanks (26 cm × 10 cm × 22 cm, length × width × height) with 3 L of water (20 cm high) was used  with a maintained water temperature with the help of heaters. The sides of the tank were visually blocked using a white opaque self-adhesive plastic film in order to reduce the influence of the surrounding area and facilitate observation. The light/dark test was performed in glass tank (20 cm × 10 cm × 8 cm, length × width × height), adapted from Rawashdeh et al.,  parted by a sliding guillotine-type partition (10 cm × 8 cm) in two equally sized dark and white compartments using black or white self-adhesive film externally covering walls, floor and the adjacent sides of the partition.  In order to allow free swim of zebrafish from one side to another the level of water in the tank was 3 cm, and the partition was raised about 1 cm above the tank floor [Figure 1].
|Figure 1: Apparatus for the study (a) general apparatus for the studies (b) apparatus to study effect of color on zebrafish movement (c) light dark test apparatus|
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Behavioral Scores in the Group Behavior Task
Height in the tank
The position (bottom × middle × upper levels) was taken as an index of anxiety, as taken in case of rodents that is, the position near the wall versus the position in center of an open field.  Fish were observed for a minute and noted according to the following scores during 1 min observations: (1) Movement restricted to the bottom third of the tank; (2) preference for the lower two-thirds of the tank; (3) similar times exploring the three thirds; (4) preference for the upper two-thirds; and 5-only in the upper third.
Locomotion was considered as a general index of behavioral excitation/inhibition. This was evaluated by comparing to "internal control" fish, taking following scores: (1) Virtually immobile; (2) slower than normal; (3) normal; (4) increased locomotion; and (5) intense locomotion.
Zebrafish show their response toward different colors. Their movement to a specific color is observed. The red region of the tank was frequently visited while the yellow region was almost neglected in normal condition while in diseased conditions this condition was reverted. Fish response to the color region was rated visually and scored as: (1) No movement to yellow region; (2) movement to yellow region 5-10 times in 1 min; (3) normal swim; (4) movement to yellow region more than 20 times in 1 min.
Zebrafish generally prefer to swim in groups and their group aggregation is termed shoal cohesion.  This behavior strategy is seen to be effective against predators in several fish species.  Shoal cohesion was measured as an individual parameter for each fish by comparing to "internal control" fish (i.e. a group of three untreated fish habituated in an independent tank) and were scored as following: (1) Complete lack of group cohesion or fish interaction; (2) loose or partial shoaling behavior; (3) normal distance and shoaling behavior compared to 'internal control'; and (4) increased shoal cohesion.
It has been that zebrafish show a marked preference for dark zones.  Based on similar results shown by rodents toward brightly illuminated areas,  this test in particular is used light/dark test is typically used for the evaluation of anxiolytics effect in rodents. Zebrafish were placed in the light zone of the apparatus with drug-free water and the following measures were recorded for 5 min: (1) Latency to the first entry in the dark compartment; (2) time spent in the light compartment; and (3) number of crossings between compartments. The apparatus was filled with 3 cm of water. This shallow tank restricts bottom-dwelling, which is a well-established anxiety behavior in a new environment. Thus, the main protective strategy is black preference, which is the measure used in this task.
The results are expressed as mean. For color affected locomotory analysis, data were analyzed with a two-tailed t-test for the preference of one color over another in each combination. The Wilcoxon matched-pairs signed-rank test was used to determine the differences between the time spent in the black and white compartments. Statistical comparison was performed to analyze time spent in light compartment (ANOVA) followed by Bonferroni's test (P < 0.05 was considered as statistically significant).
| » Result|| |
Behavioral Scores in the Group Behavior Task
Height in the tank
The height up to which the fish travelled was taken as an index of anxiety, as it is considered in the case of rodents that is, the position near the wall versus the position in center of an open field.  Results were obtained by observing the fishes for a 5 min. In case of phenytoin and gabapentin the movement was almost restricted to the bottom third of the tank for the first min while in case of VPA preference was given for the lower two-thirds of the tank. As time increased gradually, the movement shifted toward the normal conditions. In case of treatment with rutin results revealed that the movement was like the normal conditions [Figure 2].
|Figure 2: Effect of rutin on height of floating acquired during movement in zebrafish|
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Locomotion was considered as a parameter for normal behavior. During the course of treatment with antiepileptics the locomotion was increased (due to impairment) in comparison to the normal while it was toward the normal when treated with rutin which shows that rutin reverts the impairment caused due to AEDs [Figure 3].
In the previous studies, it was established that zebrafish shows response to different color. They move toward a specific color. The red region of the tank was frequently visited while the yellow region was almost neglected in normal condition while in diseased conditions this condition was reverted. When the fish were treated with AEDs, they moved in all regions while in case of treated group the response was again reverted to the normal conditions [Figure 4].
|Figure 4: Effect of rutin on locomotory effect in presence of red/yellow color on zebrafish|
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Shoal cohesion is the phenomena in which fish prefer to swim in groups, and their group aggregation is termed as Shoal Cohesion. Normally it is seen that zebrafish swim in groups. When they were treated with AEDs, a vast difference was seen in their swimming behavior. This result shows that the effects of rutin were positive over the disrupted swimming behavior produced by the administration of AEDs [Figure 5].
Treatment with AEDs caused movement of zebrafish restricted to the dark zone. Treatment with rutin caused increased latency of zebrafish to move in the light compartment. Similarly, time spent in the light compartment by zebrafish treated with Rutin was significantly (P < 0.01) more as compared to zebrafish treated with AEDs [Figure 6].
| » Discussion|| |
Zebrafish have proven to be an useful model for the study of nervous system development compared to rodent models used in pharmacological studies, zebrafish have various advantages for high-throughput screening like they are inexpensive, easier to handle due to small size, produce large numbers of progeny (up to 200 eggs in one mating), and develop rapidly (days as opposed to weeks). These beneficial characteristics of zebrafish, their external fertilization and transparency at embryonic and larval stages, makes it a useful alternative tool for the study of developing vertebrate nervous system. Even the newly developed larval zebrafish have a rich behavioral response, , thus the present study was aimed to determine the effect of rutin on brain over the adverse effects produced by AEDs using zebrafish model.
The study demonstrated that all AEDs have specifically decreased shoal cohesion and height in the tank in the GBT. The GBT task performed for these AEDs showed that the administration of these drugs has caused significant behavioral changes in locomotion, height, shoal cohesion and response to color. The light/dark task was sensitive for all antiepileptics. It is well-established that the light/dark task is an interesting screening task for antiepileptics, whereas shoal cohesion and height in the tank could serve as useful endpoints to differentiate the type of antiepileptic response. Zebrafish has a natural tendency to remain initially at the bottom of a novel environment (e.g. a test tank) and then gradually, over a few minutes, explore the higher portions of the test tank.  The fear response of zebrafish also includes forming stronger shoal cohesion, freezing and giving response to color.  The exposure to the new environment of a tank is not particularly alarming to produce such behaviors, but the effects of AEDs on shoal cohesion became apparent only after a min in the tank. These distinct time courses and sensitivities to different drugs suggest that the neurobiological systems underlying height in the tank and shoal cohesion are quite independent, but differential kinetics of the drugs tested may play a role in their observed behavioral profile. In the present study, AEDs induced an increase in the intensity response to color in zebrafish, but aggression parameters were not evaluated. The light/dark task has been classically used as an anxiety test in rodents. Anxiolytics have been found to increase time spent in the light zone whereas anxiogenic drugs decrease it. zebrafish also prefer dark environments,  which make this parameter potentially useful to assess the effects of anxiolytics. It is reported that PHT has demonstrated cognitive side effects.  These effects are dose related  and are associated with visually guided motor functions.  VPA is known to cause attentional dysfunction  along with cognitive side effects in the hyperammonemic children with epilepsy.  It is also responsible to cause reversible cortical atrophy and cognitive decline.  Similar effects are seen with levetiracetam.  However, a minimum deterioration in functioning of memory is observed. 
Our results confirmed this dark preference of zebrafish and showed that AEDs were effective in increasing time in the light zone. It should be noted that in the light/dark task the apparatus was quite different from the GBT and that fish were tested alone. These results suggest that the light/dark task may be useful for behavioral high-throughput screening of side effects produced by AEDs compounds since it is quick and easily. These results suggest that the behavioral changes produced by AEDs can be overcome by treatment with rutin.
| » References|| |
Cramer JA, Mintzer S, Wheless J, Mattson RH. Adverse effects of antiepileptic drugs: A brief overview of important issues. Expert Rev Neurother 2010;10:885-91.
Khalifa AE. Hypericum perforatum as a nootropic drug: Enhancement of retrieval memory of a passive avoidance conditioning paradigm in mice. J Ethnopharmacol 2001;76:49-57.
Kumar GP, Khanum F. Neuroprotective potential of phytochemicals. Pharmacogn Rev 2012;6:81-90.
Pyrzanowska J, Piechal A, Blecharz-Klin K, Joniec-Maciejak I, Zobel A, Widy-Tyszkiewicz E. Influence of long-term administration of rutin on spatial memory as well as the concentration of brain neurotransmitters in aged rats. Pharmacol Rep 2012;64:808-16.
Khan MM, Ahmad A, Ishrat T, Khuwaja G, Srivastawa P, Khan MB, et al.
Rutin protects the neural damage induced by transient focal ischemia in rats. Brain Res 2009;1292:123-35.
Baraban SC, Taylor MR, Castro PA, Baier H. Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 2005;131:759-68.
Berghmans S, Hunt J, Roach A, Goldsmith P. Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants. Epilepsy Res 2007;75:18-28.
Richetti SK, Blank M, Capiotti KM, Piato AL, Bogo MR, Vianna MR, et al.
Quercetin and rutin prevent scopolamine-induced memory impairment in zebrafish. Behav Brain Res 2011;217:10-5.
Piato ÂL, Capiotti KM, Tamborski AR, Oses JP, Barcellos LJ, Bogo MR, et al.
Unpredictable chronic stress model in zebrafish (Danio rerio
): Behavioral and physiological responses. Prog Neuropsychopharmacol Biol Psychiatry 2011;35:561-7.
Rawashdeh O, de Borsetti NH, Roman G, Cahill GM. Melatonin suppresses nighttime memory formation in zebrafish. Science 2007;318:1144-6.
Serra EL, Medalha CC, Mattioli R. Natural preference of zebrafish (Danio rerio
) for a dark environment. Braz J Med Biol Res 1999;32:1551-3.
Levin ED, Bencan Z, Cerutti DT. Anxiolytic effects of nicotine in zebrafish. Physiol Behav 2007;90:54-8.
Miller N, Gerlai R. Quantification of shoaling behaviour in zebrafish (Danio rerio
). Behav Brain Res 2007;184:157-66.
Gerlai R, Lahav M, Guo S, Rosenthal A. Drinks like a fish: Zebra fish (Danio rerio
) as a behavior genetic model to study alcohol effects. Pharmacol Biochem Behav 2000;67:773-82.
Bourin M, Hascoët M. The mouse light/dark box test. Eur J Pharmacol 2003;463:55-65.
Gerlai R. Zebra fish: An uncharted behavior genetic model. Behav Genet 2003;33:461-8.
Aldenkamp AP, Alpherts WC, Diepman L, van 't Slot B, Overweg J, Vermeulen J. Cognitive side-effects of phenytoin compared with carbamazepine in patients with localization-related epilepsy. Epilepsy Res 1994;19:37-43.
Gillham RA, Williams N, Wiedmann KD, Butler E, Larkin JG, Brodie MJ. Cognitive function in adult epileptic patients established on anticonvulsant monotherapy. Epilepsy Res 1990;7:219-25.
Pulliainen V, Jokelainen M. Comparing the cognitive effects of phenytoin and carbamazepine in long-term monotherapy: A two-year follow-up. Epilepsia 1995;36:1195-202.
Glauser TA, Cnaan A, Shinnar S, Hirtz DG, Dlugos D, Masur D, et al.
Ethosuximide, valproic acid, and lamotrigine in childhood absence epilepsy. N Engl J Med 2010;362:790-9.
Nicolai J, Aldenkamp AP, Huizenga JR, Teune LK, Brouwer OF. Cognitive side effects of valproic acid-induced hyperammonemia in children with epilepsy. J Clin Psychopharmacol 2007;27:221-4.
Straussberg R, Kivity S, Weitz R, Harel L, Gadoth N. Reversible cortical atrophy and cognitive decline induced by valproic acid. Eur J Paediatr Neurol 1998;2:213-8.
Meador KJ. Cognitive effects of levetiracetam versus topiramate. Epilepsy Curr 2008;8:64-5.
Helmstaedter C, Witt JA. The effects of levetiracetam on cognition: A non-interventional surveillance study. Epilepsy Behav 2008;13:642-9.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]