Bacopa monniera (L.) Wettst Ameliorates Behavioral Alterations and Oxidative Markers in Sodium Valproate Induced Autism in Rats
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- Sandhya, T., Sowjanya, J. & Veeresh, B. Neurochem Res (2012) 37: 1121. doi:10.1007/s11064-012-0717-1
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Early prenatal or post natal exposure to environmental insults such as valproic acid (VPA), thalidomide and ethanol could induce behavioral alterations similar to autistic symptoms. Bacopa monniera, a renowned plant in ayurvedic medicine is useful in several neurological disorders. The purpose of the present study was to evaluate the effect of B. monniera on VPA induced autism. On 12.5 day of gestation the female pregnant rats were divided into control and VPA treated groups. They were administered saline/VPA (600 mg/kg, i.p.) respectively and allowed to raise their own litters. Group I—male pups of saline treated mothers. On postnatal day (PND) 21 VPA induced autistic male pups were divided into two groups (n = 6); Group II—received saline and Group III—received B. monniera (300 mg/kg/p.o.) from PND 21–35. Behavioral tests (nociception, locomotor activity, exploratory activity, anxiety and social behavior) were performed in both adolescence (PND 30–40) and adulthood (PND 90–110) period. At the end of behavioral testing animals were sacrificed, brain was isolated for biochemical estimations (serotonin, glutathione, catalase and nitric oxide) and histopathological examination. Induction of autism significantly affected normal behavior, increased oxidative stress and serotonin level, altered histoarchitecture of cerebellum (decreased number of purkinje cells, neuronal degeneration and chromatolysis) when compared with normal control group. Treatment with B. monniera significantly (p < 0.05) improved behavioral alterations, decreased oxidative stress markers and restored histoarchitecture of cerebellum. In conclusion, the present study suggests that B. monniera ameliorates the autistic symptoms possibly due to its anti-anxiety, antioxidant and neuro-protective activity.
KeywordsAutismValproic acidBacopa monnieraBehavioral alterations
Bacopa monniera (L.) Wettst (Syn: Bacopa monnierri, Jal brahmi, Herpestis monniera, Gratiola monniera) (Family: Scrophularaceae) is a creeping herb found throughout India in wet, damp and marshy areas. It is well respected brain tonic in Ayurvedic medicine and best known as an intelligent nutrient, enhancing cognitive ability. Ayurvedic medicine classifies B. monniera as belonging to a group of plant medicines known as medhya rasayana that improve mental health, intellect and memory (medhya) and promote longevity and rejuvenation (rasayana) . Bacopa monniera extract has been reported to improve various neurological and neuropsychological diseases like epilepsy [1, 2], anxiety , depression , cognition or memory impairment [5, 6], cerebral injury  and exhibits very good antioxidant property [8, 9] in rat frontal cortex, striatum, and hippocampus. Clinically it has shown beneficial effect in attention deficit hyperactive disordered children . It has ability to enhance nerve impulse transmission as Bacosides aid in repair of damaged neurons by enhancing kinase activity, neuronal synthesis, and restoration of synaptic activity .
Autism is a devastating neuro developmental disorder in modern days. Its prevalence (1:150) is increasing rapidly in last 20 years that may be due to increased exposure to environmental insults such as thalidomide, ethanol, valproic acid and methyl mercury or due to increased stress during critical periods of neuronal development especially in genetically predisposed children . It is characterized by impairment in social interaction, verbal and nonverbal communication, restricted interests, and repetitive/stereotyped patterns of behavior. Sometimes it is associated with self injurious behavior and hyperactivity . Sodium valproate induced autism in rodents such as rats and mice, a well established rodent model of autism to evaluate exact pathophysiological mechanism and therapeutic drugs [13, 14]. Rats exposed to sodium valproate on 12.5 day of gestation shows several anatomical abnormalities in the brain stem, cerebellum in their offspring resembling those found in autopsy and brain imaging studies of autistic patients and exhibits behavioral abnormalities include lower sensitivity to pain and higher sensitivity to non painful stimuli, diminished acoustic prepulse inhibition, locomotor and repetitive, stereotypic like activity combined with lower exploratory activity and decreased number of social behaviors [13, 15–18]. Serotonin not only a neurotransmitter, but also regulates the development of target brain areas, such as neocortex, hippocampus, and cerebellum . Embryonic exposure to VPA in the rat also found to increase the serotonin levels in the platelets, as well as hippocampus and cerebellum . In addition, there is evidence that oxygen free radicals play a role in the pathophysiology of autism. Early prenatal or postnatal exposure to environmental toxins such as valproate, thalidomide, methylmercury and ethanol triggers the abundant release of reactive oxygen species that may be responsible for neurodevelopmental damage . Antioxidants with neuroprotective activity may show beneficial effects in autism. Vitamin E, Green tea extract was reported for protective role in sodium valproate induced autism due to its antioxidant and neuroprotective activity [21, 22]. Till today there is no specific treatment for autism, present treatment mainly aimed at symptomatic relief, there is a need to find therapeutic drugs.
However no study has been reported the effect of B. monniera on sodium valproate induced behavioral alterations and oxidative stress.
Materials and Methods
Drugs and Chemicals
Sodium valproate was purchased from Sigma Aldrich, USA. Bacopa monniera whole plant powder procured from Himalaya Co. All other reagents used in the experiment were of analytical grade purchased locally.
Female pregnant rats of 5 day old gestation were procured from National centre for laboratory animal sciences, National Institute of Nutrition, Hyderabad, India. They were housed in a room with environmentally controlled temperature (21 ± 1°C), humidity (55%) and 12-h light/dark cycle, had free access to food and water. After a 7-day acclimatization period, they were randomly selected for different experimental groups. All the experimental procedures were carried out in accordance with committee for the purpose of control and supervision of experiments on animal (CPCSEA) guidelines. The study was reviewed and approved by the Institutional Animal Ethics Committee (320/CPCSEA dated 03-01-2001). G. Pulla Reddy College of Pharmacy, Mehdipatnam, Hyderabad, India.
Preparation of B. monniera Extracts (BM)
Powdered ‘whole’ plant material of B. monniera was procured from Himalaya Drug Co., India. Plant material (25 g) was grounded to fine powder using mortar and pestle and it was then stirred vigorously in a volume of 300 ml warm distilled water for 20 min. and filtered using muslin cloth. The aqueous extract so obtained was transferred in a china dish and evaporated to dryness by heating. The extract was scraped off, weighed and reconstituted with saline before administration . The percentage yield was 18% w/w. Bacopa monniera is rich of saponins. The principal constituent present in the B. monniera extract is bacoside A. About 100 g of B. monniera extract contains ~0.576 g of Bacoside A .
Induction of Autism
On 12.5 day of gestation rats were divided into two groups. Group I (n = 12) received a single intraperitoneal injection of 600 mg/kg sodium valproate (VPA). Group II serves as control group received physiological saline. Sodium valproate was dissolved in saline at concentrations of 250 mg/ml. Administration of this dose to rats during embryogenesis has been shown to result in maximum level of total VPA (900 μg/ml) in maternal plasma in less than 1 h, with a mean plasma elimination half life of 2.3 h [13, 15–18]. Both VPA treated and control female rats were housed individually and allowed to raise their own litters. The offspring were weaned on PND 20 and rats of either sex were housed separately. Experiments were carried out on male offspring. Autism in pups was confirmed with reduced weight gain measured weekly interval, impaired olfactory discrimination on PND 9, delayed eye opening on PND 13 and14 and impaired motor development (swim performance) on PND 8 and 12 as initial activities that manifests abnormal neuron development in pups .
On PND 20 male pups were divided into three groups (n = 6). Group I (Con): Normal offspring received saline PND 21-35, Group II (VPA): Autistic offspring received normal saline from PND 21-35, Group III (VPA-BM300): Autistic offspring received aqueous extract of B. monniera (300 mg/kg/p.o.) from PND 21–35 . During the treatment period (PND 30–40) following behavioral tests were performed in the same sequence Nociception, Locomotor activity, Exploratory activity, Social behavior Anxiety and these tests were also performed in post treated period (adulthood period; PND 90–110). All the experiments were performed in the light phase between 09:00 and 15:00. At the end of experiments animals were sacrificed by cervical dislocation and brain was isolated for the biochemical estimation and histopathological studies.
Tests for Early Post Natal Development
Weight gain, eye opening, olfactory discrimination and swim performance are the tests that manifest autistic symptoms in early developmental stage . Weight gain was measured on PNDs 7, 14, 22, 50 and 90. Eye opening was observed once daily on PND 13 and 14. Olfactory discrimination test was conducted on PND 9 as VPA treated rats showed a longer latency to reach home bedding . Swim test was performed on PND 8 and 12. Swimming is a measure of motor development and integration of coordinated series of reflex responses .
Nociceptive effect was evaluated using Eddy’s Hot plate analgesiometer (INCO ltd). The hot plate which is commercially available consists of an electrically heated surface. The temperature was controlled to 55°C. The animals were placed on the hot plate and the time until either licking or jumping occurs was recorded by stop-watch .
The locomotor activity of rats was recorded individually for each animal using actophotometer (INCO pvt Ltd). Actophotometer equipped with three infrared emitters located on x- and y- axis, and with equivalent number of receivers on the opposite walls (30 × 30 × 22) (l × b×h). The locomotor activity was defined as a breakage of photo beams. Locomotor activity was assessed by comparing the number of beam breaks across a 20-min session divided into two 10-min intervals .
Social Behavior in Adolescent Period
The test area for adolescent rats consisted of an acrylic plastic circular cage measuring (25 × 20) (diameter × height) with approximately 2 cm of wood shavings covering the floor. The test cage was illuminated by 40 W red light bulb mounted 60 cm above them. Back ground noise was produced by a radio. Rats were separated and housed individually the night before the experiment to enhance later social interactions. Rats were matched for their gender and weight. Pairs of either treated or control rats were put into the apparatus over a period of 20 min. The frequency of pinning (one rat lies on its back, the other stands with two paws on top of it), following, touching, grooming each other, sniffing of any body part besides of anogenital parts and sniffing of the anogenital body parts were taken as indicators of social engagement [13, 15].
Social Behavior in Adulthood Period
Rats were separated and housed individually the night before the experiment to enhance later social interactions. The test area for adult rats consisted of plastic rectangular cage measuring (42 × 25 × 15) (l × w × h) with approximately 2 cm of husk/wood shavings covering the floor containing a plastic tube big enough for a rat to hide inside. Rats were matched for their gender and weight. Pairs of either treated or control rats were put into the apparatus over a period of 20 min. The number of pinning (one rat lies on its back, the other stands with two paws on top of it), following, touching, grooming each other, sniffing of any body part besides of anogenital parts, sniffing of the anogenital body parts and hiding inside the tube during the first and second 10 min were taken as indicators of social engagement .
Elevated Plus Maze
The elevated plus-maze was made of wood and consisted of two opposite open arms (50 × 10 cm), and two opposite arms enclosed with 40-cm high walls. The maze was elevated 50 cm above the floor. Behavior was tested in a dimly lit room with a 40 W bulb hung 60 cm above the central part of the maze. Each rat was placed for 5 min in a pretest arena (45 × 45 × 45 cm, constructed of the same material) prior to exposure to the maze. This step facilitates exploratory behavior. An investigator sitting approximately 2 m apart from the apparatus observed the rats. Immediately after the pretest, exposure rats were placed in the center of the elevated plus-maze facing one of the open arms. During the 5-min test period, the following measurements were taken: the number of entries into the open and closed arms and the time spent in the open and closed arms. From those measures, the following variables were defined: % time spent in the open arms compared to time spent in both open and enclosed arms and % open arms entries compared to both open and enclosed arms entries. An entry was defined as entering into one arm with all four paws. The maze was cleaned after each trial .
Estimation of Hippocampal Serotonin (5-HT)
Animals were sacrificed on PND 110 by cervical dislocation and brains were isolated on ice and weighed. The hippocampus was separated from the mid brain on ice. 100 mg of tissue was homogenized with 5 ml of acidified butanol (0.68 ml of 0.01 N HCl). After centrifugation for 5 min at 3,000 rpm, 2.5 ml of the supernatant was pipetted into glass tube and shaken mechanically for 5 min with 5 ml of n-heptane and 0.4 ml of 0.1 N HCl. The phases were separated by centrifugation as before. To determine 5-HT, 0.4 ml samples of the aqueous phase were pipetted into test tubes and 2.4 ml of 0.004% o-phthalaldehyde (OPT) in 10 N HCl was added. It was kept for heating in boiling water bath for 15 min. The tubes were cooled in water and fluorescence was measured using spectrofluorometer. Activation and fluorescent wavelengths were 360 and 470 nm respectively. Blanks were prepared by reacting 0.6 ml of the OPT solution with 0.1 N HCl. Standards were prepared as 60 μg/ml in deionized water, diluted 1:100 for use with 0.1 N HCl containing and 0.1 ml reacted with 0.6 ml of 0.004% OPT in HCl solution [23, 24].
Estimation of Oxidative Parameters
Estimation of Total Nitrite Levels
Nitrate/nitrite level estimation is a measure of nitric oxide produced. The frozen brain tissue was weighed and homogenized in 50 mmol/L potassium phosphate buffer (pH 7.8) and centrifuged at 11,000g for 15 min at 4°c. 200 μl of supernatant was mixed with 200 μl saturated solution of vanadium trichloride in 1 N HCl and immediately 200 μl Griess reagent (0.1% N-(1-naphthyl) ethylene diamide dihydrochloride, 1% sulfanilamide in 5% phosphoric acid) was added and mixed it, incubated at 37°C for 30 min and the absorbance was measured at 540 nm using auto analyser. The concentration was calculated from standard curve of potassium nitrite and expressed as micromoles of nitrate/nitrite [18, 25, 26].
Estimation of Reduced Glutathione Level (GSH)
Reduced glutathione (GSH) level was measured in brain using the method described by Ellman (1959) . The homogenate (in 0.1 M phosphate buffer, pH 7.4) was added with equal volume of 20% trichloro acetic acid containing 1 mM EDTA to precipitate the tissue proteins. The mixture was allowed to stand for 5 min prior to centrifugation for 10 min at 200 rpm. The supernatant (200 μl) was then transferred to a new set of test tubes and added 1.8 ml of the Ellman’s reagent (5, 5′ -dithio bis-2-nitro benzoic acid) (0.1 mM) was prepared in 0.3 M phosphate buffer with 1% sodium citrate solution). Then all the test tubes make up the volume of 2 ml. After completion of the total reaction, solutions were measured at 412 nm against blank. The GSH concentration in the sample is calculated using the molar extinction coefficient of TNB (tri nitro benzoic acid) 14,150 M−1Cm−1 and expressed as μmol/L tissue homogenate .
Estimation of Catalase
Immediately after sacrificial of animals, the brain was dissected and homogenized in 5 ml of 0.05 M phosphate buffer, pH 7.0; used for catalase determination. Catalase activity was measured by method that employs H2O2 to generate H2O and O2. The activity was measured by degree of this reaction. The standard substrate contained 0.3 ml of H2O2 in 50 ml of 0.05 M phosphate buffer pH 7.0. The sample aliquot (20μl) was added to 980μl of substrate mixture. After 1 min, initial absorbance was added and final absorbance was read after 6 min. This reaction was taken at 230 nm .
Brain was isolated immediately after sacrifice of animal and placed in 10% neutral formalin solution, processed and embedded in paraffin. Sagittal sections of cerebellum (5 μm thick) were stained with haemotoxylin and eosin (H&E) and analyzed using a light microscope for changes in Purkinje cell layer and neuron structure. Photographs of sections were taken using camera .
Data expressed as mean ± SEM were analyzed by one way analysis of variance (ANOVA) followed by Newman-Keuls test as a post hoc to compare more than two groups and student ‘t’ test was used for comparison between two groups by using Graph Pad prism software (5.0 version). p values less than 0.05 were considered as statistically significant.
Administration of VPA on 12.5 day of gestation induces autistic behavior manifested by following:
Tests for Early Post Natal Development
Reduced weight gain, delayed eye opening, impaired olfactory discrimination and swim performance indicates the autistic symptoms in VPA treated pups (data not shown).
Effect of B. monniera Extract on Thermal Nociception in VPA Exposed Rats
Effect of B. monniera Extract on Locomotor Activity in VPA Treated Rats
Effect of B. monniera Extract on Exploratory Activity in VPA Treated Rats
Effect of B. monniera Extract on Anxiety in VPA Treated Rats
Effect of B. monniera Extract on Social Behavior in VPA Treated Rats
Effect of B. monniera on Serotonin Levels in Brain of VPA Exposed Rats
Effect of Bacopa monniera on Hippocampal serotonin, reduced glutathione, catalase, and total nitrite in rats prenatally exposed to VPA
Hippocampal serotonin (μg/g of tissue)
0.4417 ± 0.034
0.9168 ± 0.109**
0.717 ± 0.115
Reduced glutathione (μM/L)
39.66 ± 1.608
31.47 ± 1.494**
37.21 ± 1.563+
141.9 ± 6.405
97.87 ± 3.973**
136.4 ± 10.75++
72.55 ± 3.587
107.3 ± 7.349**
88.68 ± 4.526+
Effect of B. monniera on Total Nitrite, Reduced Glutathione (GSH) and Catalase in Brain of VPA Exposed Rats
Induction of autism significantly (p < 0.01) increased total nitrite levels and decreased GSH, catalase levels compared to control group. Treatment with B. monniera significantly reversed the total nitrite (p < 0.05), GSH (p < 0.05) and catalase (p < 0.01) levels in brain compared to VPA exposed rats (Table 1).
The present study demonstrated the protective effect of B. monniera on VPA induced behavioral alterations, serotonin levels, and oxidative parameters. Exposure to VPA on 12.5 day of gestation, the time of neural tube closure and development of first neurons, has shown long term effects on post natal behavior in male rats . In agreement with previous reports in the present study administration of VPA on 12.5 day of gestation developed autism manifested by lowered body weight gain, impaired olfactory discrimination, reduced swim performance, delayed eye opening and behavioral aberrations such as decreased sensitivity to pain, increased locomotor activity in novel environment, associated with decreased exploratory activity, increased anxiety in elevated plus maze, decreased social explorations in both adolescence and adulthood period [13, 15, 16, 30].
Prenatal exposure to VPA evokes changes in nociception which persist till adulthood. In accordance with previous reports we observed increased pain threshold in VPA treated rats compared to control animals [13, 15, 30]. It is suggested that VPA exposure during neuronal development might cause dysregulation of enkephalinergic system and proenkephalin levels . It is also suggested, that may be due to increased GABA levels. Elevated GABA levels in early developmental period may result in dysregulation of GABAergic system in later development . Bacopa monniera significantly reduced pain threshold in adulthood period and adolescence period of VPA treated rats which could be due to its neuroprotective activity that may protects sensory neurons affected by VPA exposure .
In agreement with previous reports in our study VPA exposed animals was shown increased number of locomotor behavior in novel environment, anxiety in elevated plus maze [13, 15–18]. This possibly due to brain anomalies induced by VPA or might be due to increased glutaminergic transmission leading to neural hyper excitability in brain regions [16, 32]. In the present study we observed that B. monniera normalized the locomotor activity and anxiety in VPA exposed rats. Protective effect of B. monniera on locomotor activity may be due to its anti anxiety activity , and it decreases accumulated glutamate . Decreased exploration in VPA rats suggests that the effect mediated rather by fear related inhibition of exploratory activity or decreased motivation to explore a novel environment and abnormal fear conditioning in VPA rats with non goal directed locomotor activity may suppress exploration [15–17]. Treatment with B. monniera normalized exploratory activity in VPA rats suggests efficacy of this drug on prevention of anxiety .
In agreement with previous results VPA rats had shown attenuated social behavior (number of pinnings, grooming, followings, climbings, sniffings and anogenital sniffings) may be due to decreased ability to express and understand intra specific communicative signals and to perform social behaviors in adequate sequences [13, 15–18]. It may be as a consequence of elevated anxiety and fear which may have profound influence on these behaviors in rats . Treatment with B. monniera increased the social behavior in VPA rats.
In consistent with previous reports VPA rats had shown increased serotonin levels in the hippocampus [20, 33]. Early abnormality of the 5-HT system might be followed by post natal abnormalities such as abnormal 5-HT concentration and abnormal neural network formation . In our study B. monniera treatment partially decreased hippocampal serotonin levels. It may attribute to up-regulated serotonin transporter (SERT) expression that enhances the reuptake of released serotonin [35, 36].
Involvement of oxidative stress is one of the causative factors for autism. Pre or post natal exposure to environmental pro-oxidants such as valproic acid may trigger the formation of reactive oxygen species. Reactive oxygen species will interfere with the neurodevelopmental process or cause neural damage which may results in abnormal behavioral process. It is suggested that oxidative stress is the mechanism that associating the environmental exposure to toxicants during critical periods cause genetic alterations especially in genetically susceptible children . In the present study increased total nitrite and decreased glutathione and catalase levels indicate involvement of oxidative stress in VPA induced autism. Treatment with B. monniera significantly reversed the altered oxidative stress markers due to its antioxidant property in rat brain [8, 9, 38].
Our histopathological findings suggests reduced number of purkinje cells in VPA treated group and it has also shown neuronal degeneration and chromatolysis that indicate VPA induced apoptosis in neurons which is in consistent with previous reports [29, 39]. Bacopa monniera treatment improved the VPA induced histopathological changes in cerebellum possibly due to its neuroprotective effect [2, 38, 40].
On the basis of these findings, we conclude that B. monniera ameliorates VPA induced behavioral deficits, increased serotonin, altered oxidative stress markers and histopathological findings may due to its anti anxiety, antioxidant and neuroprotective activity.
Summary and Conclusion
In the present study administration of VPA on 12.5 day of gestation significantly induced autism manifested by long term behavioral alterations in both adolescence and adulthood period, altered antioxidant profile and serotonin level. On other hand induction of autism significantly showed histopathological findings in cerebellum characterized by decreased Purkinje cell distribution, neuronal degeneration and chromatolysis. Treatment with B. monniera significantly improved behavioral alterations in two developmental periods, ameliorated oxidative stress markers and histopathological findings.
In conclusion, the present study suggests that aqueous extract of B. monniera may beneficial in treatment of autism. This protective effect of B. monniera may attribute to the anti-anxiety, antioxidant and neuroprotective activity.
We express our sincere gratitude towards Dr. B. Madhava Reddy Garu, Principal, G. Pulla Reddy College of Pharmacy, Hyderabad for his keen interest and support during the course of the study and we thank Dr. A. Anand kumar, Department of pathology, College of veterinary science, Sri Venkateshwara university, Hyderabad for his support in technological aspect.