(MWM) Test

SUPPLEMENTAL TEXT

MATERIALS AND METHODS:

Morris Water Maze (MWM) Test: MWM test to assess spatial reference memory was performed as per published protocol (Vorhees and Williams, 2006). Briefly, the animals were trained to learn spatial location of a hidden platform in a circular pool (210 cm in diameter, 53 cm in height with non reflective interior surface) filled with water at 25 ± 2ºC. During the training regimen of 5 days, each rat was allowed 4 sessions per day (total 20 sessions) with an inter-trial interval of 5 min. A different starting point was used in each of the 4 trials. The trial consisted of gently placing the rat into water, facing wall of the pool and allowing it to locate the submerged platform. The rat was allowed to remain on the platform for 30 sec and then placed back into its home cage after wiping it completely. In the event of rat failing to locate the platform within 60 sec, it was guided to the platform manually. For recording specific parameters during the experiments, the tank was divided into four imaginary quadrants, namely, South East (SE or Q1), North East (NE or Q2), North West (NW or Q3) and South West (SW or Q4). The platform was placed in the center of Q1 quadrant and the location was maintained throughout the experiment. An overhead camera coupled to a computer-assisted tracking system, ANY-Maze (Stoelting, USA) was used to record the position of rat in the pool. The parameters recorded during the experiments included Latency (time taken to reach the platform; measured in seconds), Path Length (distance travelled by rats to find the platform; measured in meters), Entry Point and Quadrant-specific Latency. Bar graphs were plotted utilizing Mean ± S.D data. A probe trial (PT) was performed at the end of 5 days

training and prior to specific exposures. For the same, the platform was removed from target quadrant and various parameters were recorded. The time taken by the animal to first hit the target area (where platform was placed during training) was recorded as PT latency and distance covered to reach it as PT Path length. These were used as ‘animal-specific reference values’ for calculating percentage change in Latency and Path length parameters of individual animals, after specific (hypoxic/Normoxic) exposures. This data was represented as box-whisker plots (see results). For all experiments, the significance of inter-group differences was evaluated by oneway analyses of variance (one-way ANOVA). p-value < 0.05 was considered to be statistically significant. Bonferroni multiple comparison tests were performed as a post hoc analysis to compare differences between the groups, whichever found significant. All statistical analyses were done using GraphPadInStat version 3.00 for Windows (GraphPad Software, San Diego, California, USA).

T-Maze Test: For alternative test of cognition, ‘Rewarded Alternation Test’ was performed employing TMaze, as per protocol described in Deacon and Rawlins, 2006. Briefly, the animals were habituated to maze instrument such that the animals ran freely in this area (4 days). The reward pellet was also fed in home cages of the animals to habituate them to its taste. The usual feed was maintained at 80% of animals’ free-feeding weight. Subsequently, the animals were trained for 5 consecutive days, with each animal receiving 10 trials per session (each day) at regular intervals (around 10 animals received trials in succession before the first animal took trial again). In each trial, ‘Sample phase’ consisted of animals running from start arm to goal arm, with the other arm blocked by its doors. In the ‘Choice phase’, both arms were opened. The retention time of 60

seconds was allowed between sample and choice phase. To avoid any possible bias, an independent observer, blinded to all groups, recorded the observations. ANOVA was used for multiple group comparison.

Intracellular Cleaved-Caspase 3 Staining, TUNEL Assay, Flow Cytometry: The hippocampal tissue was isolated from 3 animals (for each group), pooled (group-wise) and processed to generate cell suspension, utilizing NeuroCult™ Enzymatic Dissociation Kit for Adult CNS Tissue (Cat. No. 05715, STEMCELL Technologies Inc, Vancouver, Canada) as per manufacturer’s protocol. Subsequently, intracellular cleaved-Caspase 3 (active form) staining was performed utilizing BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (Cat. No. 554714) and apoptotic cells were detected employing ‘Flow Cytometry kit (TUNEL assay) for Apoptosis’, Sigma Aldrich. The cells were finally re-suspended in Propidium Iodide containing buffer before acquisition using Flow Cytometer (FACS Calibur, BD). In both these assays, population was first gated on FL2H (Propidium Iodide positive) to remove any noncellular tissue debris and subsequently analyzed for fluorescence patterns on FL1H (Alexa Fluor 488/FITC labeled targets, anti-cleaved-Caspase 3 or anti-BrdU antibodies respectively, in individual experiments), as shown in the figures.

In Situ TUNEL assay: Cell death was assessed by in situ labelling of DNA fragments utilizing TUNEL assay kit (ApopTag Fluorescein in situ apoptosis detection kit, Millipore, USA). The fluorescent signal was visualized at an appropriate excitation/emission wavelength and the positive cells were

scored at a suitable magnification utilizing a digital camera attached to the microscope (Olympus, BX51TF).

RNA isolation: The animals were sacrificed and the brain tissues were isolated quickly. Hippocampus was dissected carefully and snap frozen at -80ºC. The tissues samples from five animals were pooled for each group and the total RNA was isolated utilizing TRI Reagent (Sigma Aldrich) as per manufacturer’s instructions. The integrity of RNA was checked on 0.8% agarose gel (with formaldehyde) and was further purified for gene expression experiments employing RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNase I digestion.

Microarray analysis: One-color microarray based gene expression analysis was performed utilizing Agilent microarray platform. Briefly, purified total RNA (40 ng) was subjected to two consecutive rounds of T7-promoter based linear amplification to generate labeled complementary RNA. mRNA labelling, hybridization and washing steps were carried out as per manufacturer’s instructions using the Quick-Amp labeling Kit (p/n5190-0442) and in situ Hybridization Kit (5188-5242) respectively, from Agilent Technologies. Quality of RNA was checked by Bioanalyzer and quantified using Nanodrop (Agilent Technologies) prior to hybridization to GE 8 X 60K whole rat genome microarray gene expression chips (Agilent Technologies) following manufacturer’s protocol. The microarray chips were washed and scanned immediately using a Microarray Scanner (Model G2565BA, Agilent Technologies). The images were verified manually and ensured to be devoid of any uneven hybridization, streaks, blobs and other

artifacts. Data pre-processing and differential expression analysis was conducted by R software using Bioconductor packages as reported previously (Sharma et al., 2014). The data were background corrected and normalized between arrays using quantile method in order to compensate for any systematic technical differences between chips. Differential expression analysis was carried out utilizing the linear modelling features of the Bioconductor LIMMA package, which fits a linear model to the expression value for each gene and assesses the significance of differential expression between different experimental conditions. The analysis involved estimating consistent, closed form estimators for the hyper parameters using marginal distributions of selected statistics. ‘Robust Linear Method’ (RLM), was adopted for estimating model coefficients, as it is a strong tool to identify outliers, measure errors and other data irregularities. These estimators were based on the two-step weighted least squares method, where weights were adaptively computed using the empirical distribution of residuals obtained from initial robust fit. The Benjamini and Hochberg’s method was used to control false discovery.

Weighted Gene Co-Expression Network Analysis (WGCNA): R package was used for executing WGCNA as described in Langfelder and Horvath, 2008. A matrix consisting of log2 values of fold changes of transcripts differentially expressed (by at least 2 fold) at one of the time points (Day 1, 3 and 7) was generated utilizing R package and used as input for WGCNA. The correlation matrix was obtained by calculating the Pearson’s correlation between differentially expressed genes, with specific values across the time points. The ‘pickSoftThreshold’ function was used to transform the matrix into a signed adjacency matrix network. We chose '13' as the soft-threshold power. Topological overlap (TO), a biologically meaningful measure of node similarity, was calculated. The genes were hierarchically clustered

using 1-TO as the distance measure and modules were determined by using a dynamic treecutting algorithm. The Module Eigengene (ME, the first principal component of expression values) was calculated using all genes in each identified module. The ‘mergeCloseModules’ function was used to merge modules in gene expression networks that were too close as measured by the correlation of their Eigengenes. Further, the Eigengenes were re-calculated for the merged modules. Eigengenes of all modules were correlated with the measured value of Path Length and Latency in MWM test to identify modules that were significantly associated with the changes in spatial memory. The modules were characterized using BiNGO and over-represented groups of GO terms/functional domains were identified by hypergeometric test using Benjamin & Hochberg False Discovery Rate (FDR) correction and a p-value threshold of 0.01. In addition, the relationships between modules were represented as a heat map and the corresponding module Eigengene values were shown as a bar plot.

Transmission Electron Microscopy: Animals were anesthetized with ketamine (50 mg per kg body weight, i.p.) and xylazine (10 mg per kg body weight, i.p.). Transcardiac perfusion using a peristaltic pump was carried out using cold PBS. The hippocampus was dissected and sectioned into 2-mm-thick coronal blocks, which were fixed overnight with 2.5% glutaraldehyde and 2% paraformaldehyde in phosphate buffer at 4°C, washed in 0.1 M phosphate buffer, pH 7.4 and then post-fixed for 15 mins in 1% osmium tetroxide prepared in the same buffer. The specimens were dehydrated through a graded series of ethanol, exchanged through propylene oxide, and embedded in araldite CY. Ultrathin hippocampal sections were obtained using ultra microtome (Ultracut UCT; Leica, Australia) with a diamond knife. The sections (1 mm × 1 mm) were then stained with uranyl acetate and lead

citrate followed by imaging under a Morgagni 268D (Fei Company, The Netherlands) transmission electron microscope.

Gelatin Zymography: Protein extracts from the hippocampal tissues were prepared by sonication in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, Protease inhibitor cocktail containing 104 mM AEBSF, 80 µM Aprotinin, 4 mM Bestatin, 1.4 mM E-64, 2 mM Leupeptin and 1.5 mM Pepstatin A (Sigma Aldrich)). The protein concentration was estimated using Bicinchoninic Acid assay Kit (BCA; Pierce Biotechnology). A total of 60 µg of protein was mixed with an equal volume of 2X sample buffer (62.5 mM Tris-HCl (pH 6.8), 20% Glycerol, 4% SDS, 0.01% Bromophenol Blue) and subjected to electrophoresis on 10% polyacrylamide gels containing gelatin (1 mg/ml) under non-reducing conditions. The gels were washed with 2.5% Triton X-100 and incubated in digestion buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.06% Brij-45) at 37°C for 24 hrs. The gels were subsequently stained in 0.25% Coomassie Blue R-250 solution followed by destaining in solution containing methanol/glacial acetic acid/water. As required, MMP inhibitors, o-phenanthroline and EDTA were added in the digestion buffer during the 24 h incubation.

Western Blotting: 30 µg of total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Protran, Sigma Aldrich). The blots were blocked in 5% non-fat dry milk and probed with desired primary antibody. Anti-Caspase 3, anti-ICAM-1, anti-Tubulin, anti-CBS, anti-β-Actin and anti-GFAP were purchased from Abcam.

Horseradish peroxidase-conjugated secondary antibody (Sigma Aldrich) was used along with Enhanced-Chemiluminescent (ECL) Kit (Sigma Aldrich) for detection.

Histological analysis: Rats were anesthetized with ketamine (50 mg per kg body weight, i.p.) and xylazine (10 mg per kg body weight, i.p.) and transcardially perfused with PBS followed by ice-cold 4% paraformaldehyde prepared in PBS. Brain tissues were quickly dissected, post-fixed in 4% paraformaldehyde prepared in PBS for 24 h and cryoprotected in successive 20% and 30% sucrose solution (in 0.1 M phosphate buffer, pH 7.4). 30 µm thick sections, containing the hippocampal region, were cut using a cryostat and processed as described below.

Immunohistochemistry: Sections were washed in PBST (PBS containing 0.1% Tween-20) and heat induced antigen retrieval was carried out with 10 mM sodium citrate buffer (pH 6.0) for 15 min. The sections were blocked at 25°C for 2 hrs with 5% goat serum prepared in PBS and 0.03% Triton X-100. After three washes with PBST, the sections were incubated overnight at 4ºC with appropriate primary antibody (anti-Caspase 3, anti-vWF, anti-CD31, Abcam). HRP-conjugated secondary antibody (Sigma Aldrich) was used for detection employing 3,3′-diaminobenzidine (DAB). The sections were rinsed with distilled water, dehydrated in ethanol and mounted onto gelatin-coated slides using DPX mounting medium (Sigma Aldrich). Images were then captured with a digital camera attached to the light microscope (Olympus BX 51TF). The expression of specific target was calculated by intensity thresholding method utilizing Image J software, NIH.

Immunofluorescence: Sections were processed as described for immunohistochemistry experiments. For coimmunofluorescence experiments; labeled primary antibody, single (anti-GFAP) or pooled (antiLaminin with anti-Aqp-4 & anti-CD31 with anti-IgG, Abcam) were utilized. The sections were counter stained with DAPI (4′, 6-diamidino-2-phenylindole; 1:1000, from 2mg/ml stock, Sigma Aldrich) and mounted using ProLongAntifade kit (Invitrogen, USA). Confocal microscopy was carried out and images were acquired on a Leica TCS SP5 AOBS scanning laser microscope using oil immersion objective at desired magnifications. Co-localization was estimated by calculating Pearson’s Correlation between various channels, utilizing Image J software, NIH.

NOx and cGMP Estimation: NOx and cGMP measurements were performed utilizing commercially available kits from Cell BioLabs Inc [OxiSelect™ In Vitro Nitric Oxide (Nitrite /Nitrate) Assay Kit (Fluorometric), Cat. No. STA-801] and Enzo Life Sciences Inc [cGMP ELISA Kit; Cat. No. ADI-900-013] respectively, as per manufacturer’s protocols.

Estimation of Sulfide levels by Zinc Precipitation Assay: Total free sulfide estimation in tissue samples was done as described in Ang et al., 2012. Briefly, the brain tissues were homogenized in 50 mM sodium carbonate buffer (pH 9) at a concentration of 40 mg/ml. 500 µl of homogenate was added to a pre-mixed solution of 1% zinc acetate (350 µL) and 50 µL of 1.5 M NaOH. The tubes were then centrifuged at 5000 rpm, 4ºC for 12 mins to pellet zinc sulfide (white precipitate). The supernatant was carefully removed and the pellet was washed with water by constant vortexing, followed by centrifugation at 5000 rpm,

4ºC for 12 mins. Supernatant was then removed and 160 µl of water was added to reconstitute the pellet. 40 µL of 1:1 pre-mixed dye containing 20 mM N, N-dpd, prepared in 7.2 M HCl and 30 mM FeCl3 prepared in 1.2 M HCl was added to dissolve the pellet. The solution was then incubated at room temperature for 10 min and absorbance was recorded at 670 nm. Spectral scans from 500-700 nm were also recorded to observe λmax. All solutions used for estimation were prepared in degassed water.

Cerebral Blood Flow Measurement: Cerebral Blood Flow (CBF) was estimated utilizing Laser Doppler Flowmetry, as per published protocol (Sutherland et al., 2014). Briefly, the skull was exposed in anesthetized animals and thinned to translucency utilizing a stereotaxic micro motor high-speed drill (Stoelting, USA) over a fixed region [Bregma coordinates AP: −3.0 mm; L: −7.0 mm] established utilizing motorized stereotaxic set up (Stoelting, USA). A needle probe TSD144 (BIOPAC Inc, USA) was stably positioned over the drilled area, utilizing needle probe holder. CBF was recorded utilizing LDF100C (Laser Doppler blood perfusion monitor, BIOPAC Inc, USA) for 3-5 minutes with stable readings. The mean blood perfusion units (BPU) were calculated utilizing AcqKnowledge software (BIOPAC).

Whisker Stimulation: Whisker stimulation was achieved by manually displacing the whiskers (uncut) on right side, utilizing a paintbrush, in a rhythmic manner (2-3 Hz) for 10 minutes, in anesthetized animals. The CBF was recorded over a fixed point in contralateral barrel cortex [Bregma coordinates AP: −3.0 mm; L: −7.0 mm], before and after whisker stimulation, utilizing Laser Doppler Flowmetry,

as described above. For each group (N, H, HD, ND), data from a minimum of 5 animals was recorded. The difference in CBF, before and after whisker stimulation (∆BPU), was calculated and mean ± S.D for each group was plotted. One-way ANOVA was utilized for testing statistical significance of the data set. * P < 0.05.

REFERENCES Ang, A.D.K., A; Giles, G.I; Bhatia, M (2012). Measuring free tissue sulfide. Advances in Biological Chemistry 2, 360-365. Deacon, R.M., and Rawlins, J.N. (2006). T-maze alternation in the rodent. Nature protocols 1, 712. Langfelder, P., and Horvath, S. (2008). WGCNA: an R package for weighted correlation network analysis. BMC bioinformatics 9, 559. Sharma, M., Singh, S.B., and Sarkar, S. (2014). Genome wide expression analysis suggests perturbation of vascular homeostasis during high altitude pulmonary edema. PloS one 9, e85902. Sutherland, B.A., Rabie, T., and Buchan, A.M. (2014). Laser Doppler flowmetry to measure changes in cerebral blood flow. Methods Mol Biol 1135, 237-248. Vorhees, C.V., and Williams, M.T. (2006). Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nature protocols 1, 848-858.

LEGENDS TO SUPPLEMENTAL FIGURES

Supplemental Figure 1: Networks of Genes related to non-redundant biological processes significantly enriched from differentially expressed genes at day 3; post HH, in Hippocampus. The list of differentially expressed genes (Hippocampus, day 3 post HH) was subjected to analysis employing ‘GeneMANIA’ and the networks representing significantly enriched biological processes were represented as 'Degree sorted circular view'. Supplemental Figure 2: Networks of Genes related to non-redundant biological processes significantly enriched from differentially expressed genes at day 7; post HH, in Hippocampus. The list of differentially expressed genes (Hippocampus, day 7 post HH) was subjected to analysis employing ‘GeneMANIA’ and the networks representing significantly enriched biological processes were represented as 'Degree sorted circular view'.

LEGENDS TO SUPPLEMENTAL TABLES Supplemental Table 1: List of differentially expressed genes (compared to Normoxic Controls), as inferred from microarray analysis of RNA from hippocampus, after 1 day of exposure to Hypobaric Hypoxia. Gene Names, Accession Numbers and respective Fold change (Log2) are shown along with other details as indicated. Supplemental Table 2: List of differentially expressed genes (compared to Normoxic Controls), as inferred from microarray analysis of RNA from hippocampus, after 3 days of exposure to Hypobaric Hypoxia. Gene Names, Accession Numbers and respective Fold change (Log2) are shown along with other details as indicated. Supplemental Table 3: List of differentially expressed genes (compared to Normoxic Controls), as inferred from microarray analysis of RNA from hippocampus, after 7 days of exposure to Hypobaric Hypoxia. Gene Names, Accession Numbers and respective Fold change (Log2) are shown along with other details as indicated. Supplemental Table 4: List of Panther and KEGG pathways significantly enriched from differentially expressed genes at day 3; post Hypobaric Hypoxia, in Hippocampus. The differentially expressed genes at Day 3 post HH, in Hippocampus, were subjected to analysis employing DAVID Bioinformatics resource and list of Panther and KEGG pathways significantly enriched from this data set along with Gene count, percentage, p-Value, Gene Symbols and respective Fold Change (Log2 values) is represented. Supplemental Table 5: List of Panther and KEGG pathways significantly enriched from differentially expressed genes at day 7; post Hypobaric Hypoxia, in Hippocampus. The differentially expressed genes at Day 7 post HH, in Hippocampus, were subjected to analysis employing DAVID Bioinformatics resource and list of Panther and KEGG pathways significantly enriched from this data set along with Gene count, percentage, p-Value, Gene Symbols and respective Fold Change (Log2 values) is represented.

SUPPLEMENTAL FIGURE 1

HIPPOCAMPUS DAY 3 NETWORKS

EXTRACELLULAR MATRIX

REGULATION OF CELL ADHESION

ELEVATION OF CALCIUM ION CONCENTRATION

CIRCULATORY SYSTEM PROCESS

REGULATION OF ANGIOGENESIS

CELL CELL JUNCTION

COAGULATION

REGULATION OF BEHAVIOUR

NEURON PROJECTION DEVELOPMENT

REGULATION OF MAPK

LEUKOCYTE MIGRATION

MUSCLE CONTRACTION

HOMEOSTASIS

SUPPLEMENTAL FIGURE 2 REGULATION OF CELL ADHESION

RESPONSE TO OXYGEN LEVEL

CIRCULATORY SYSTEM PROCESS

DENDRITIC SPINE HEAD

HIPPOCAMPUS DAY 7 NETWORKS EXTRACELLULAR MATRIX

RESPONSE TO COTICOSTEROID STIMULUS

CALCIUM ION HOMEOSTASIS

REGULATION OF NERVE TRANSMISSION

ANGIOGENESIS

SMOOTH CELL PROLIFERATION

REGULATION OF NEUROLOGICAL SYSTEM

G PROTEIN SIGNALLING