Computational protocol: Early Life Stress Enhancement of Limbic Epileptogenesis in Adult Rats: Mechanistic Insights

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Protocol publication

[…] To label new born cells in the DG, rats were administered 7 daily injections of BrdU (5-bromo-2-deoxyuridine, 50 mg/kg ip dissolved in saline) (Sigma St Louis, MO, USA) beginning on the final day of kindling after the 5th Class V seizure. Two weeks after the last BrdU injection, rats were transcardially perfused with 4% paraformaldehyde, brains removed and frozen in cryoprotectant. Serial coronal sections (20 µm) encompassing the hippocampus were cut on a cryostat, slide-mounted and stored at −80°C until further use.The staining protocol was adapted from previous studies , , , . Briefly, sections were washed with 0.1 M TBS (Tris buffer saline, pH 7.4) and microwave antigen retrieval performed in a 0.1 M citrate buffer at pH 6.0. Sections were rinsed in 1% hydrogen peroxide in 50% methanol, and, for BrdU staining only, incubated in 2 N HCl (37°C) to denature DNA, then neutralized with 0.1 M sodium borate (pH 8.5). Sections were incubated overnight at 4°C with primary antisera: monoclonal rat anti-BrdU (Accurate Chemical, Westbury, NY; 1∶400), monoclonal mouse anti-NeuN (Chemicon, Temecula, CA; 1∶200), or monoclonal mouse anti-synaptophysin (Sigma, USA; 1∶200), all diluted in 0.3% Triton X100 in 0.1 M TBS. This was followed by 2 hours incubation in appropriate biotinylated secondary antibodies: goat anti-rat (Vector Laboratories, Burlingame, CA, USA; 1∶400), goat anti-mouse (Vector Laboratories, Burlingame, CA, USA; 1∶200), all diluted in 4% NGS, followed by incubation in avidin-biotin-peroxidase (Vectastain ABC-Kit, Vector Laboratories), and then diaminobenzidine (MP Biomedicals, Solon, OH, USA). These sections were used for localising BrdU+ve cells, counting of CA3c neurons, and assessing synaptophysin expression, as appropriate. Digital images of synaptophysin-stained sections were taken at 100× magnification and relative optical density (ROD) was measured in the CA3c region using ImageJ software (NIH) . [...] All quantitative histological analyses were performed on coded slides with the investigator blind to treatment group. For statistical testing we utilised Statistica® software (Tulsa, OK). Afterdischarge thresholds were analysed using one-tailed Student's t-test. The numbers of stimulations required to reach each of the 5 stages of kindling were compared between the groups using one-way ANOVA with repeated measures followed by planned comparisons. CA3c neuronal numbers and BrdU+ve cell numbers were analysed with two-way ANOVA, with early life interventions and kindling as independent variables and, if a significant effect was found, this was followed by planned comparisons to compare for differences between specific groups. Proportions of BrdU+ve cells co-localizing with NeuN in kindled rats were analysed using the Chi-square test. CORT responses were compared using ANOVA with repeated measures (time), and, if appropriate, a planned comparison at each time point. Data were analysed separately for each sex. Subsequently, sex differences were analysed using a three-way ANOVA with sex, kindling and early life interventions as the independent variables. The data are presented as mean ± SEM unless otherwise stated. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus, Homo sapiens
Diseases Epilepsy, Temporal Lobe, Hypothalamic Neoplasms, Fractures, Stress
Chemicals Bromodeoxyuridine, Corticosterone