INTRODUCTION:

Autism is the most severe condition among a group of autism spectrum disorders (ASDs) that also includes Asperger syndrome, pervasive developmental disorder not otherwise specified, and rare syndromic forms such as fragile X and Rett syndromes. Autism typically presents before 3 years of age, the prevalence of ASDs is estimated in children, with a male-female ratio of 4:1 [1]. Sodium valproate (VPA) is a commonly used antiepileptic drug. In utero exposure to VPA increases the risk for neural tube defects in humans [2, 3] and rats [4].

Prenatal VPA exposure has been shown to affect behavioral development with treated rats exhibiting repetitive/stereotypic-like behavior, decreased exploratory activity and a decreased number of social behaviors [5, 6]. Abnormalities of the hippocampus and related limbic structures have been hypothesized to be relevant to the pathophysiology of autism because of their role in learning, emotion and functions that are typically disturbed in autism. These observations lend credibility to the suggestion that early exposure to VPA in rats leads to behavioral and neurological deficits that resemble autism [7]. Polyphenols are a large and diverse class of compounds, many of which occur naturally in a range of food plants. The flavonoids are the largest and best-studied group of these. A range of plant polyphenols are either being actively developed or currently sold as dietary supplements and/or herbal remedies [8]. Tea is one of the most widely consumed beverages in the world. The increasing health benefits of tea have led to the inclusion of tea extracts in dietary supplements and functional foods. Green tea (non-oxidized), Oolong tea (partially oxidized), and Black tea (oxidized), three major categories of tea obtained from the plant Camellia sinensis (L.) Kuntze, belonging to the family Theaceae, differ in terms of their manufacturing and chemical composition [9,10]. The major flavonoids of green tea are catechins, which include (-)-epicatechin (EC), (-)-epicatechin-3-gallate (ECG), (-)-(EGC) and (-)-epigallocatechin-3-gallate (EGCG), (+)-catechin (C), (+)-gallocatechin (GC), (+)-catechin gallate (CG), and (+)-gallocatechin gallate (GCG), [11,12]. Green tea also contains gallic acid, other phenolic acids such as chlorogenic acid and caffeic acid. Flavonols such as kaempferol, myricetin and quercetin are the other bioconstituents of green tea [13]. The current study has reported to establish the impact of EGCG on behavioral regressions in rats treated postnatally with VPA.

MATERIALS AND METHODS:

Chemicals and Reagents:

VPA and EGCG were purchased from Sigma‑Aldrich (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade obtained from Himedia laboratory Ltd., Mumbai, India. EGCG was administered orally by every day at the dose of 2 mg/kg body weight (b.w.)

Induction of Autism:

All the animals (Wistar rats) were fed with the standard pellet diet (Hindustan Lever Ltd., Bangalore, India) and water was made available ad libitum. Food and water were replenished daily. The animals were housed in polycarbonate cages in a room with 12 hours day-night cycle, temperature of 22±2°C and humidity of 45–64%. The experimental protocol was approved by the committee for research and animal ethics, Esma institute of technology laboratory, Karur (Vide No: DIC (2008/33/014/00049/ BEYA/TN/1695(BSS)/2014) and were in accordance with the guidelines of Indian Council of Medical Research (ICMR) [14].On the 12^th day of gestation, female rats were divided into two groups.

Group I:

Treated: Received a single intraperitoneal injection of VPA 600mg/kg (n =12).

Group II:

Control: Received physiological saline (n =12).

VPA was dissolved in saline at concentrations of 250mg/ml [4]. Both VPA treated and control female rats were housed individually and allowed to raise their own litters. The offspring is weaned on postnatal day (PND 20) and rats of either sex housed separately. Experiments are carried out only on male offspring.

Experimental Design:

On PND 20, male pups were divided into six groups (n=6), Group I (Control): Normal offspring received saline PND 21-90, Group II (EGCG 2mg/kg): Normal offspring received EGCG PND 21-90, Group III (VPA): Autistic offspring received normal saline from PND 21-90, Group IV (VPA+EGCG 2mg/kg): Autistic offspring received EGCG from PND 21-90. Behavioral studies have carried out in experimental animals.

Exploratory Activity:

The exploratory activity was assessed using Hole board apparatus (45×45×45) (l×b×h). Number of rearing and hole-poking (nose of the animal put inside the hole) were measured during a 3 min time session. Background noise was produced by a radio [15, 16].

Open-field Behavior Test:

Open-field behavior is a simple test to evaluate the status of the animal by placing the animal in a brightly lit rectangular box (100 x 100 cm) with 40 cm height, made with walls of plywood. The floor consists of a clean plastic material painted in black dividing the field into 25 (5 x 5) equal squares. The rats are placed in the corner of the rectangular box and their behavior is observed for five minutes. This elicits a series of behavior-like peripheral and central movements and motor activity like grooming and rearing, related to the emotional status of the animal [17].

Traversing Beam Test:

Trained animals were allowed to walk on a narrow flat stationary wooden beam (L 100 cm × W 1 cm) placed at a height of 100 cm from the floor. The time taken to cross the beam from one end to the other was counted as described earlier. Animals on the beam were videotaped to analyze stepping errors during the experiment [17].

Statistical Analysis:

The data were subjected to one-way analysis of variance followed by Duncan’s multiple range test (DMRT) using SPSS software 12.0. Results were expressed as mean ±SD for six rats in each group. P values <0.05 were considered significant.

RESULTS AND DISCUSSION:

Induction of autism in VPA rats significantly decreased peripheral and central movements on PND 90 when compared with the control group. Open field are paradigms employed to screen anti-anxiety agents. Postnatal treatment with VPA is believed to evoke anxiety and exaggerated fear identical to that seen in autistic subjects [15, 18]. Treatment with EGCG produced a significant increase in time spent and number of open arms entries (peripheral and central movements); suggesting that fear and anxiety are substantially decreased. Evaluation of the pattern of exploration and horizontal distance traveled in an open field following treatment with EGCG suggests that stereotypic forms of locomotion were significantly controlled VPA induced damage to the neuroanatomy of the brain could alter ancillary and associated functions were shown in Fig 1 and 2. Induction of autism significantly decreased rearing, grooming and hole poking behavior on PND 90 when compared to control group. 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 [16,19,20]. Treatment with EGCG reversed the altered exploratory activity, grooming and rearing in both developmental periods when compared to VPA exposed rats were shown in Fig 3-6 A and B.

Fig.1 Peripheral movement’s activities were measured during the 5 min trial in the open field test for control and experimental rats. Values are expressed as mean± SD (one-way ANOVA followed by DMRT). VPA p<0.05 compared with control rats, VPA+EGCG p<0.05 compared with VPA exposed rats.

Fig.2 Central movement’s activities were measured during the 5 min trial in the open field test for control and experimental rats. Values are expressed as mean± SD (one-way ANOVA followed by DMRT). VPA p<0.05 compared with control rats, VPA+EGCG p<0.05 compared with VPA exposed rats.

Fig.3 The Grooming activities were measured during the 5 min trial in the open field test for control and experimental rats. Values are expressed as mean± SD (one-way ANOVA followed by DMRT). VPA p<0.05 compared with control rats, VPA+EGCG p<0.05 compared with VPA exposed rats.

Fig.4 The rearing behavioral were measured during the 5 min trial in the open field test for control and experimental rats. Values are expressed as mean± SD (one-way ANOVA followed by DMRT). VPA p<0.05 compared with control rats, VPA+EGCG p<0.05 compared with VPA exposed rats.

Fig.5 The exploratory behaviors were measured in control and experimental rats for 5 min. Values are expressed as mean± SD (one-way ANOVA followed by DMRT). VPA p<0.05 compared with control rats, VPA+EGCG p<0.05 compared with VPA exposed rats.

Fig.6 A and B. The beam walking and foot slip performance were measured in control and experimental rats. More time taken to traverse the beam with higher number of foot slip errors were noticed in VPA rats compared with control rats. Prior treatment with EGCG significantly reduced beam crossing duration and foot slip errors in autistic rats. Values are expressed as mean± SD (one-way ANOVA followed by DMRT). VPA p<0.05 compared with control rats, VPA+EGCG p<0.05 compared with VPA exposed rats.

CONCLUSION:

In utero exposure to VPA early in development resulted in behavioral abnormalities. Our behavioral study supports the findings of developmental changes in neural tube closure in rats. In this respect, EGCG may have a beneficial effect in reversing behavioral abnormalities in autistic rats due to the anti-anxiety, antioxidant and neuro protective activity.

CONFLICT OF INTEREST:

Hereby, the authors declare that they have no conflict of interest.

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Received on 02.04.2017 Modified on 27.04.2017

Research J. Pharm. and Tech. 2017; 10(5): 1477-1480.

DOI: 10.5958/0974-360X.2017.00260.8