Computational protocol: Chronic Early-life Stress in Rat Pups Alters Basal Corticosterone, Intestinal Permeability, and Fecal Microbiota at Weaning: Influence of Sex

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Protocol publication

[…] Feces were collected from PND21 male and female rats for DNA extraction and amplification of the V4 region of the 16S rRNA gene. Genomic DNA was extracted using the Powersoil kit as per manufacturer’s instructions (MoBio Laboratories Inc, Carlsbad, CA, USA). The V4 region of 16S ribosomal RNA genes was amplified and underwent paired end sequencing on an Illumina MiSeq using the 2 × 150 bp v2 kit as previously described. The 253 base pair reads were processed using QIIME v1.9.1 with default parameters. Observable taxonomic units (OTUs) were picked against the May 2013 version of the Greengenes database, pre-filtered at 97% identity. Sequence depth ranged from 75 603 to 110 767. Alpha diversity metrics (ie, bacterial diversity within a sample) and beta diversity (differences in composition across samples) were calculated in QIIME v1.9.1 using OTU-level data rarefied to 75 603 sequences. Alpha diversity metrics included Faith’s phylogenetic diversity metric, Chaol, and Shannon index. [...] Data are expressed as mean ± SEM and were analyzed using GraphPad Prism 4 software (GraphPad, San Diego, CA, USA). Plasma corticosterone in CTL and LNS groups were compared by Student’s t test. Pearson’s correlation coefficient was used to assess the correlation between corticosterone plasma levels and adrenals weights/100 g body weight and between corticosterone plasma levels and Akkermansia abundance. Correlation analysis between corticosteronemia and adrenal weights were only reported in female rats as no significant change in adrenal weights was observed between male pups exposed or not to LNS procedure. One-way ANOVA followed by Tukey post hoc test comparisons were used to analyze in vivo intestinal permeability. Interaction between treatment and sex was analyzed by two-way ANOVA followed by Bonferroni post hoc test. A P-value < 0.05 was considered significant. The significance of differences in alpha diversity was calculated using the Mann-Whitney U test. Beta diversity was calculated using unweighted UniFrac and visualized by principal coordinates analysis. Adonis, a permutational analysis of variance, was performed using 100 000 permutations to test for differences in beta diversity between the LNS and control groups. Association of microbial genera and OTUs with LNS or control groups were evaluated using DESeq2 in R. Unrarefied 16S rRNA count data was first filtered to remove OTUs present in only one sample then fitted to multivariate negative binomial models with LNS group and sex as covariates. P-values for differential abundance were converted to q-values to correct for multiple hypothesis testing (P < 0.05 for significance). DESeq2 models were also run with intestinal permeability or plasma corticosterone as covariates in addition to LNS group and sex to identify taxa associated with these parameters. […]

Pipeline specifications

Software tools QIIME, UniFrac, DESeq2
Databases Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Rattus norvegicus
Diseases Drug Hypersensitivity, Hypothalamic Neoplasms, Fractures, Stress, Irritable Bowel Syndrome
Chemicals Corticosterone