2. MD natur Laboratories (Hammamet, Tunisia) as

2. Materials and methods: 2.

1. Plant material and extracts preparation:Commercial Harpagophytum procumbens (Devil’s claw, DC) powdered tubers was provided by MD natur Laboratories (Hammamet, Tunisia) as capsules containing dried coarse powder. The plant material was ground to a fine powder, passing through a mesh to obtain a uniform granulometry.

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The obtained powder was protected from light and stored at 4°C until use. Then, 500 mg of DC roots powder were resuspended in 10 ml of distilled water and leaved to macerate for 3 hours at 4°C. The suspension was filtered through a 100 ?m mesh. The extract was freshly prepared the day of experiment. Lipidium sativum (Garden cress, GC) seeds were collected from a local market (Souk El Blat) in the medina of Tunis.

Seeds were identified and authenticated by Dr. Mouhiba NASRI AYACHI, professor of plant taxonomy at the faculty of sciences, University of Tunis El Manar, Tunisia. The plant material was stored at room temperature in a dry place prior to use. Then, 3 g of fine powder of seeds were extracted by maceration in 10 ml of distilled water at ambient temperature for 3 h with continuous stirring. The suspension was filtrated with No.

1 Whatmann Millipore filter paper. The extract was freshly prepared one day before treatment. 2.

2. Animals and experimental design: The study was conducted according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal handling and procedures were carried out under strict compliance with guidelines for Ethical control and supervision in the Care and Use of animals. Males (300-350 g) and female (200-250 g) Wistar rats were purchased from the Tunisian society of Pharmaceutical Industries of Tunisia (SIPHAT) and allowed to acclimatize for two weeks prior to mating. All animals were housed (2-3 per cage) under photoperiod cycle (12-h: light: 12-h dark; lights on at 7:00 am) in a room with controlled temperature (24 ± 1°C) and humidity (50 ± 10 %).

Food (standard rodent pellets provided from El Badr Society, Bizerte, Tunisia) and water were available ad libitum. After acclimatization, non-gravid female rats were mated with males (Female: male = 2:1) and vaginal smears were checked daily. The day of positive-sperm detection in the vaginal smear was considered as a gestational day 0 (GD 0). A total number of 40 pregnant dams were singly-housed in propylene cages and randomly divided into two groups (n=20 per group): the first group was orally given equal amount of vehicle (corn oil) and used as the control, the second received a dose of 500 ?g/ kg of BW/day of DEHP (Sigma, purity 99 %, CAS # 117-81-7) dissolved in corn oil.

Female rats were treated from gestational day 1 to day 21 of lactation. Following weaning, at PND 1, the female pups were culled and males were used for the rest of the experiment. 24 male pups from each group were housed 2 per cage (in same previous housing conditions) and were administered the same as before (control group receiving corn oil and DEHP group receiving 500 ?g/kg of BW/day of DEHP) until 9-months age (adulthood). DEHP solutions were prepared freshly before each treatment.

The dose of DEHP was chosen because high doses were used previously in many research papers to evaluate the adverse effects of DEHP exposure and here we investigate the neurotoxic effect of a low environmentally relevant-dose. At the end of DEHP exposure, rats were assigned to 4 groups of 12 animals each. Control group was divided into two groups: Group I (Vehicle Sham) receiving 1 ml of corn oil daily and Group II (DcR sham) receiving an intragastrically aqueous extract of Devil claw’s dried roots at a dose of 50mg/kg of BW/day.

Rats previously treated with DEHP were also divided into two groups: Group III (DEHP): which continued to receive corn oil and Group IV (DEHP-DcR) consisted of DEHP-intoxicated rats which were given 50 mg/kg of BW/day of Devil claw’s dried roots aqueous extract. The experimental protocol is summarized in figure x. All groups were treated under the same housing conditions for a period of 30 days. Animals were weighted and behavioral observations were scored daily until the end of the experiment.

2.3. Behavioral tasks: 2.3.1. Forced swim test (FST): The forced swim test (FST) is one of the most commonly used assays for the study of depressive-like behavior in rodents (Slattery and Cryan, 2012).

The FST was performed according to a previously reported method described by Yankelevitch-Yahav et al. (2015). The test consists of two swimming sessions: an initial 15-min pretest followed, 24 hours later, by a 5-min test. Briefly, swimming sessions were conducted by placing the rats in a transparent cylindrical Plexiglas container (50 cm in height x 20 cm diameter) filled with tap water at 25°C. The water in the cylinder was changed after each tested rat. The test session was video-recorded from the front of the cylinder for later scoring.

The 5-min test was scored for the total duration of immobility (floating with the absence of any movement except for those necessary to keep the nose above the water), struggling/climbing (tentative to escape) and swimming (Movement of forelimbs and hind limbs around the cylinder). 2.3.2. Crawley’s sociability and preference for social novelty test: The three-chamber paradigm known as Crawley’s sociability and preference for social novelty test is employed to asses social affiliation and social memory in rodent models of central nervous system disorders.

The social approach apparatus is comprised a rectangular box made of glass and divided into three chambers (25 cm length x 20 cm width x 50 cm Hight) with two clear glass walls allowing free access into each chamber. The wire box containers were made of cylindrical chrome bars spaced 1 cm apart. The test was carried out as described previously by Kaidanovich-Beilin et al.

(2011). Briefly, during the habituation phase, each of the two side chambers contained an empty wire box container, the subject rat was placed at the center of the middle chamber to habituate for 5 minutes. During the sociability phase, an unfamiliar rat (Stanger 1) was enclosed inside a wire box in one of the side chambers, the experimental rat was allowed to freely explore each of the three chambers for 10 minutes. The placement of the novel rat (Stranger 1) in the left or right-side chambers altered across a test rat. During the social novelty session, a new unfamiliar rat (Stranger2) was enclosed in the box container that had been empty during the sociability phase and we proceeded in the same way as the social affiliation session. Exploration of a stranger rat or an empty box container was defined as when the tested rat oriented toward the box container with a distance between the nose and the box less than 1 cm, or as climbing on the box.

The time spent in each chamber and time spent exploring empty box container vs box housing stranger 1 (in sociability phase) or between experimental rat and the box housing Stranger 1 vs Stanger 2 (in social novelty preference phase) was recorded from a camera mounted in front of the test apparatus. 2.3.3.

Elevated plus maze (EPM): The EPM test of anxiety is an elevated “+” shaped apparatus consisted of 4 arms (each 50 long x 10 cm wide) and elevated 80 cm above the floor. Two opposed arms have 40 cm high dark walls and are considered as “closed arms”. The other two arms have no walls and are considered as the “open arms”.

All arms are joined by a 10 cm x 10 cm wide central part. The test was conducted according to the method described by Li et al. (2015). Rats were individually placed in the center of the maze facing an open arm to avoid direct entrance into a closed arm, left to freely choose for an open or close arm and allowed for 5 minutes free-exploration. After each trial, the apparatus was cleaned with 75% ethanol to prevent olfactory cues and to ensure proper disinfection.

The time spent in each arm and the number of entries were video-recorded then scored. Entrance in one arm is defined as having all, four paws inside the arm. Anxious rats were more likely to enter and to stay in the closed arms. 2.

3.4. Successive alleys test: The successive alleys test of anxiety is a modified version of the elevated plus maze (Barkus et al., 2010). The apparatus consists of four successive alleys, connected in a serial manner, of growing anxiogenic character (By gradually decreasing the width of alleys and the height of the side walls and by increasing the brightness of each alley (from black (Alley 1) through grey (Alley 2) to white (Alleys 3 and 4)) the apparatus was made of painted wood and was elevated 50 cm above the floor. Construction details for successive alleys apparatus are summarized in table x.

The test was performed as previously described by Deacon (2013). Briefly, Rats were placed at the closed end of alley 1, facing the end wall. The amount of time spent and the number of entries into in each alley were video-recorded and scored during total test time of 5 minutes. The apparatus was cleaned with 75% ethanol between each tested rat to remove olfactory cues. Time spent on, and entries into, the lighted open alleys were considered an inverse index for anxiety-like behavior: the lower is this index, the more anxious the rat is. 2.

3.5. Open field (OF) test: The open field test is another widely used behavioral assay to evaluate anxiety-like behavior and locomotor responses to novel environments in rodents (Klarer et al., 2014). The apparatus was a 90 x 90 x 45 cm3 wooden box.

For data analysis, the arena was divided into 36 equal squares. The squares along the side are considered as peripheral squares and the other as central squares. This paradigm is based on the natural preference of rodents to be near protective walls rather than exposed to hazard out in the center (Christakis et al., 2012). Each rat was gently placed in the center of the OF arena and allowed to freely explore for 5 min. Behavior was recorded using a video camera mounted directly above the apparatus.

The indices including the time spent in the central quadrants (considered as an inverse index of anxiety-like behavior), the number of square crossing (counted as an index of locomotor activity) rearing and grooming numbers (assessed for stereotypic behavior) and fecal grains were measured. The OF apparatus was cleaned with 75% ethanol after each tested rat in order to reduce odor interference. 2.3.

6. Dark-light test: Anxiety-like behavior was assessed on….day after the end of treatment, in the light-dark task as previously described (Christakis et al., 2012). The light-dark apparatus is an enclosed rectangle, divided into two compartments, one which was made of translucent glass (light lit compartment; 40 x 40 x 40 cm3; 50 lux) and the other which was painted black (dimly lit compartment; 40 x 40 x 40 cm3; 5 lux). The compartments were connected via a small doorway (5 x 10 cm2) allowing transition between the two sides.

Rats were individually placed into the light side and allowed to freely explore for 5 minutes. The test was videotaped and the number of transitions between the two compartments and the time spent in each side were manually scored using the recorded videos. Urine and feces were cleaned with 75% ethanol after each test session to eliminate odor cues. • Christakis, D.

A., Ramirez, J. S., ; Ramirez, J. M.

(2012). Overstimulation of newborn mice leads to behavioral differences and deficits in cognitive performance. Scientific reports, 2, 546.

• Klarer M, Arnold M, Günther L, Winter C, Langhans W, Meyer U. Gut vagal afferents differentially modulate innate anxiety and learned fear. J Neurosci. 2014 May 21;34(21):7067-76.• Slattery DA, Cryan JF. Using the rat forced swim test to assess antidepressant-like activity in rodents.

Send to Nat Protoc. 2012 May 3;7(6):1009-14. doi: 10.1038/nprot.2012.044.

• Yankelevitch-Yahav, R., Franko, M., Huly, A., Doron, R. The Forced Swim Test as a Model of Depressive-like Behavior. J.

Vis. Exp. (97), e52587, doi:10.

3791/52587 (2015).• Kaidanovich-Beilin, O., Lipina, T., Vukobradovic, I., Roder, J.

, Woodgett, J.R. Assessment of Social Interaction Behaviors. J.

Vis. Exp. (48), e2473, doi:10.3791/2473 (2011).• Barkus C, McHugh SB, Sprengel R, Seeburg PH, Rawlins JN, Bannerman DM. Hippocampal NMDA receptors and anxiety: at the interface between cognition and emotion.

Eur J Pharmacol. 2010 Jan 10;626(1):49-56.• Deacon, R.M.

The Successive Alleys Test of Anxiety in Mice and Rats. J. Vis. Exp.

(76), e2705, doi:10.3791/2705 (2013).• Li C, Liu Y, Yin S, Lu C, Liu D, Jiang H, Pan F.

Long-term effects of early adolescent stress: dysregulation ofhypothalamic–pituitary–adrenal axis and central corticotropinreleasing factor receptor 1 expression in adult male rats. Behav Brain Res. 2015 Jul 15;288:39-49.

doi: 10.1016/j.bbr.2015.

04.007. Epub 2015 Apr 13.

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