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  • All the experimental protocol and animal care was approved by the ethical committee of the Defence Institute of Physiology and Allied Sciences (27/1999/CPCSEA) in accordance with the guidelines of “Committee for the Purpose of Control and Supervision of Experiments on Animals” of Govt. of India. All surgery was performed under sodium pentobarbital anesthesia and all efforts were made to minimize the suffering to the animals. Three month old male Sprague Dawley rats (230 g) were used in the present study which were procured from animal house facility of the Institute. They were maintained in a temperature controlled room (25±2°C at 65±5% humidity) on a 12/12 h light/dark cycle with food and water available ad libitum. Rats were tested for anxiety levels in a plus maze and categorized into anxious and normal individuals based on their preference for closed and open arms. The normal individuals were then taken for further experimental purpose. Rats (n = 200) were divided into different experimental groups (Table 1) for further investigation. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0062235.t001 Shows experimental design describing groups, number of individuals, exposure duration and interventions. Rats were randomly assigned to the enriched or the standard environmental condition. Enriched environment cages consisted of clear Plexiglas cages (35 inch ×20 inch ×25 inch) with two horizontal platform (20×15 inch) dividing the cage into three floors. On the ground floor, there was a plastic running wheel, nesting material and an assortment of differently colored and textured plastic toys (balls, tubes, boxes and bells) that were changed every 2 days. Steel ladders allowed rats to reach the upper floors, where they had access to food and water. Standard cages were clear Plexiglas laboratory cages (18×25×13 inch). Rats were kept in standard cage in groups of 3 each whereas in enriched cage they kept in group of 6 each. Enriched and standard cages were placed in a temperature-controlled room (22°C) with a light–dark 12∶12 cycle (light on 0700–1900 hrs). Food and water were given ad libitum. Rats were housed in each experimental condition for 7 days. To determine the role of neurotrophins, intracerebroventricular (ICV) injection of either vehicle (VEH; PBS, pH 7.4) or tyrosine kinase (Trk) inhibitor i.e., K252a was administered (250 µM, 5 µl) into the left hemisphere of adult rat brain. The Trk gene silencing was performed in vivo by administering TrkA and TrkB antisense oligonucleotides before HH exposure to rats. The antisense TrkB sequence refers to the sequence from nucleotide 651 to 670 (5′ ACTGGCAGCTCGGGATGTCG-3′). The antisense nucleotide, complementary to that sequence was 5′- CGACATCCCGAGCTGCCAGT-3′. The antisense TrkA sequence (59- AACTGTTGTTGTGTCC-39) corresponds to nucleotides 623–638 of rat TrkA. A random oligonucleotide (59- GTAAATTGACCAGGAG-39) was used as a control. The oligonucleotides were dissolved in a buffer containing10 mM Tris-Cl, pH 7.2, and 1 mM EDTA to a final concentration of 200 mM. Antisense oligonucleotides and randomized controls have been designed and manufactured by Biognostik (Gottingen, Germany). Intracerebroventricular (ICV) injection of either vehicle (VEH; PBS, pH 7.4) or antisense was administered into the left hemisphere of adult rat's brain as described in supplementary information (Supplementary Information File S1). An ERK1/2 inhibitor, U0216; a PI3 kinase inhibitor, wortmannin and GSK3ß inhibitor, AR-A014418 were purchased from Sigma Aldrich (San Diago, CA). U0216 and Wortmannin were dissolved in PBS whereas AR-A014418 was dissolved in DMSO and administered before HH exposure at a concentration of 1nmol, 0.1nmol and 30 µmol respectively [18]. Rats were ICV injected with a 5 µl solution containing either VEH (1 µl of DMSO and 4 µl of PBS, pH 7.4) or inhibitor as stated above. Following probe trial the rats were divided into different groups as shown in Table 1. The hypoxic and hypoxic with enriched environment groups were exposed to a simulated altitude of 7600 m (25,000 ft) in a specially designed hypobaric chamber where altitude could be maintained by reducing the ambient barometric pressure. The exposure was for continuous 7 days with a 15 min interval each day during the exposure for replenishment of food and water and changing the cages of the animals. Fresh air was continuously flushed at a rate of 8 L/min to prevent accumulation of carbon dioxide within the chamber. The temperature and humidity in the chamber were maintained at 28±2°C and 60±3% respectively. The rate of ascent and descent was maintained at 300 m/min. Reference memory was assessed in Morris Water Maze [25]–[26] to investigate spatial learning and memory of rodents. Before HH exposure animals were trained for 8 days and probe trial was performed before and after HH exposure. Single task of memory test was performed after exposure to HH. Detailed protocol for training, probe trial and memory task are as follows. Prior to exposure to hypobaric hypoxia rats were trained in a Morris water maze to study the effect of enriched environment on spatial reference memory acquisition. The maze comprised of a white circular pool of 180 cm diameter, filled with water (temperature around 24–25°C, depth 50 cm). The pool was conceptually divided into four equal quadrants having 4 points designated as starting positions (North, South, East or West). The pool had an escape platform submerged in water (2 cm below the water surface) which was camouflaged by mixing non toxic white paint to the water. The rats were painted black using a hair dye as the software detected the movements based on a phase contrast principle. The position of a black rat in the white maze was recorded by an overhead camera and computer assisted tracking system (Columbus instruments, USA). The animals were divided into different groups and trained for 8th days (sessions) and a probe trial was performed on the 9th day. Each session consisted of four trials with a 15 min inter-trial interval. The platform position remained in the same location throughout the training period. A trial began when the rat was released into water facing the wall of the pool at one of the 4 starting positions that was chosen at random. The order of the starting position varied in every trial and any given sequence was not repeated in the next session. During the trial, the rat was allowed to locate the submerged platform for 60 s. If the animal failed to reach the platform it was gently guided to it and left on it for 10 s. The latency (in seconds) and pathlength (in cm) to find the platform was recorded in each trial and mean latency and pathlength for each session was calculated. Twenty four hours after the end of the last training on the eighth day, rats were subjected to probe trial, a task, which was designed to assess the consolidation and retrieval of reference memory. It consisted of a single trial wherein the rats were allowed to swim for 60 s, with the platform removed from its position. Here, the number of crossing over at the original platform position and the time (s) spent in the target quadrant were measured. Rats were found to spend maximum time in the target quadrant and the crossing over frequency averaged around 5 during the probe trail indicating consolidation of memory. (iii) Memory test (testing for retention of learned task): Rats in all the groups were immediately tested for memory retention following the stipulated period of 7 days exposure to hypobaric hypoxia for all groups. The rats were subjecting to a probe trial followed by a single trial of the task that was given during training for acquisition of spatial reference memory. The time spent and distance traveled to reach the platform, time spent in the target quadrant, and number of crossing over at the platform position was recorded during the probe trail. After exposure and memory assessment rats were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p. ), perfused transcardially with chilled PBS and decapitated. The brain were dissected out and used for different experimental protocols accordingly. Neuronal death by Cresyl Violet Staining: After hypoxic exposure, rats were anesthetized and perfused with ice cold PBS, fixed with 4% paraformaldehyde, cryoprotected in 30% sucrose solution, sliced into 10 µm free floating sections and processed for Cresyl violet staining. Morphology of neurons in CA1 regions was studied. Briefly every sixth section from each individual was taken, dehydrated using gradient alcohol concentration, stained with cresyl violet for 3 mins and then rehydrated again. The sections were transferred to slides and mounted with permanent mounting media. Morphology of neurons in CA1 region was observed under light microscopy. Counting of pyknotic/dead cells was done using Stereo investigator (MBF Biosciences, USA). Neuronal degeneration was assessed histologically in each of the experimental groups by fluoro jade (FJ) B staining method as described previously [27]. Briefly, every sixth section from each individual was taken for assessment of neuronal degeneration in CA1 region in hippocampus. The sections were stained according to the protocol suggested by the manufacturers and sections were visualized at 480 nm excitation and 525 nm emission using a fluorescent microscope (OLYMPUS) with FITC filters. The total number of fluoro jade positive cells were scored in each of the regions and results were depicted in terms of percentage increase in fluoro jade positive cells taking average number of fluoro jade positive cells in normoxic group to be 100%. RNA isolation and PCR array: Total RNA was isolated from rat hippocampus using a Qiagen total RNA isolation kit according to the manufacturer's recommendations. Rat signal transduction pathway RT2 Profiler PCR Array and RT2 Real-Time SyBR Green/ROX PCR Mix were purchased from Super Array Bioscience (Frederick, MD). PCR was performed on ABI Step One Plus (Applied Biosystems), according to the manufacturer's instructions. Samples from all groups were compared. For data analysis, the ΔΔCt method was used with the aid of a Microsoft excel spreadsheet containing algorithms provided by the manufacturer. Fold-changes were then calculated and expressed as log-normalized ratios of values from all tissues. Samples from each group (50 µg) were loaded onto SDS-PAGE gels. Following electrophoresis gels were transferred by semi dry transblot (Biorad) to nitrocellulose membranes and probed against rabbit polyclonal AKT, pAKT, mouse monoclonal ERK, pERK and rabbit monoclonal GSK3ß, pGSK3ß, CREB and pCREB antibody (Sigma, St. Louis, Mo., USA) at a concentration 1∶1000, and further with respective secondary antibody labelled with horseradish-peroxidase at a concentration 1∶5000 (Santa Cruz, CA). The membranes were developed using the enhanced chemiluminescence light (ECL) (Sigma. USA) and then membrane was exposed to X-ray film for detection. The Asp-Glu-Val-Asp-(7-amino-4-methylcoumarin) (DEVD-AMC) cleavage assay was performed as described previously (Jain et al.,2012). Acetyl-AMC (Calbiochem) was used to obtain a standard curve, and the enzyme activity was calculated as picomoles of AMC/mg of protein/min. The data of 8 day acquisition trials in Morris Water Maze was averaged for each session of four trials. The performance during trials was analyzed using two-way Analysis of Variance (ANOVA) with trials as repetitive measures. In the probe trial task (single trial), at the end of 8 days training and again after exposure to hypoxia platform crossings, time spent in target quadrant in initial 10 s, was analyzed using one-way ANOVA. Similarly, mean of latencies and pathlength for reference memory testing after exposure to hypobaric hypoxia was analyzed in similar manner since a single trial was given to each animal during the task. The results of histology and all biochemical estimations are representations of 6 individual observations and statistical analysis for multiple comparisons was done using two-way ANOVA. Data is presented as mean ± SEM. Pair wise comparisons were used to evaluate the difference between different experimental groups. All analysis was followed by post hoc student's Newman's Keul's test, when necessary. Probability value ≤5% (p<0.05) was considered significant for all statistical analysis.
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