Maternal separation prior to neonatal hypoxia-ischemia: Impact on emotional aspects of behavior and markers of synaptic plasticity in hippocampus

Exposure to early-life stress is associated with long-term alterations in brain and behavior, and may aggravate the outcome of neurological insults. This study aimed at investigating the possible interaction between maternal separation, a model of early stress, and subsequent neonatal hypoxia-ischemia on emotional behavior and markers of synaptic plasticity in hippocampus. Therefore, rat pups (N = 60) were maternally separated for a prolonged (MS 180min) or a brief (MS 15min) period during the first six postnatal days, while a control group was left undisturbed. Hypoxia-ischemia was applied to a subgroup of each rearing condition on postnatal day 7. Emotional behavior was examined at three months of age and included assessments of anxiety (elevated plus maze), depression-like behavior (forced swimming) and spontaneous exploration (open field). Synaptic plasticity was evaluated based on BDNF and synaptophysin expression in CA3 and dentate gyrus hippocampal regions. We found that neonatal hypoxia-ischemia caused increased levels of anxiety, depression-like behavior and locomotor activity (ambulation). Higher anxiety levels were also seen in maternally separated rats (MS180min) compared to non-maternally separated rats, but prolonged maternal separation prior to HI did not potentiate the HI-associated effect. No differences among the three rearing conditions were found regarding depression-like behavior or ambulation. Immunohistochemical evaluation of synaptophysin revealed that both prolonged maternal separation (MS180min) and neonatal hypoxia-ischemia significantly reduced its expression in the CA3 and dentate gyrus. Decreases in synaptophysin expression in these areas were not exacerbated in rats that were maternally separated for a prolonged period prior to HI. Regarding BDNF expression, we found a significant decrease in immunoreactivity only in the hypoxic-ischemic rats that were subjected to the prolonged maternal separation paradigm. The above findings suggest that early-life stress prior to neonatal hypoxia-ischemia leads to significant alterations in synaptic plasticity of the dorsal hippocampus during adulthood, but does not exacerbate HI-related changes in emotional behavior

Additional file 1: of Humoral response in experimental autoimmune encephalomyelitis targets neural precursor cells in the central nervous system of naive rodents

Methods. Experimental groups, EAE induction and evaluation, Cell culture protocol, Preparation of samples for SDS-PAGE. (RTF 3.45 kb

Additional file 1: Figure S1. of Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination

Bielschowsky staining, immunofluorescence, and real-time PCR quality controls. (A1–4) Typical images of scores 0–3 in Bielschowsky silver staining. (B, C) Preabsorption assay for LINGO-1 and TROY in serial sections of chronic and acute phases, respectively (the phase where signal is most abundant). (B1) LINGO-1 antibody specifications: rabbit polyclonal, Abcam ab23631, LOT N/A, dilution 1:300. (B2) LINGO-1 peptide specifications: rabbit, Abcam ab25890, LOT #GR41007-1, incubation with ab in 10× molecular ratio. (C1) TROY antibody specifications: goat polyclonal, Santa Cruz sc-13711 (E-19), LOT #H0707, epitope mapping near the C-terminus of TROY of mouse origin, dilution 1:100. (C2) TROY peptide specifications: goat, Santa Cruz sc-13711 P, LOT #B0402, incubation with ab in 10× molecular ratio. (D, E) Antibody specificity test for p75 and NgR with the use of another antibody (different company) recognizing a different epitope in serial sections of chronic and acute phases, respectively (the phase where signal is most abundant). (D1) p75 antibody #1 specifications: mouse monoclonal, Abcam ab8877, LOT GR136825-1, ME20.4, dilution 1:400. (D2) p75 antibody #2 specifications: mouse monoclonal, Santa Cruz p75 (B-1) sc-271708, LOT #J0611, epitope mapping between amino acids 393–427 at the C-terminus of NGFR p75 of human origin, 1:100. (E1) NgR antibody #1 specifications: rabbit polyclonal, Santa Cruz sc-25659 (H-120), LOT E1209, epitope corresponding to amino acids 31–150 mapping near the N-terminus of Nogo-R of human origin, dilution 1:100. (E2) NgR antibody #2 specifications: rabbit polyclonal, Abcam ab26291, LOT N/A, epitope from within residues 150–250 of rat Nogo receptor, dilution 1:100. (F) β-actin real-time PCR quality control showing the specific amplification products on agarose gel and the melting curves of their respective genes; curve identifier: light green TROY, orange p75, dark green LINGO-1. (G) mRNA levels of coreceptors LINGO-1, p75, and TROY in the spinal cord of EAE animals by real-time PCR analysis using GAPDH as a second, quality control, house-keeping gene. The levels of mRNA expression for the coreceptors (G1) LINGO-1, (G2) p75, and (G3) TROY, followed the same pattern with those that underwent β-actin analysis. Error bars indicate the standard statistical error of the mean (SEM), ***p < 0.001, **p < 0.01. Black scale bar = 20 μm

Additional file 2: Figure S1. of Humoral response in experimental autoimmune encephalomyelitis targets neural precursor cells in the central nervous system of naive rodents

Evidence of the production of anti-MOG-immunoglobulins within antisera when mice were inoculated with MOG peptide. (A) Purified EAE-AS (IgG from EAE-AS) and unpurified EAE-AS identified bands of the same molecular weight on NPCs substrate. Western blot of recombinant MOG as SDS-PAGE substrate (B) and ICC of EL4-MOG cells (C) revealed the real existence of anti-MOG-immunoglobulins within EAE-AS. One band was yielded (above 20kDA) when recombinant MOG was probed with EAE-AS and anti-MOG (positive control) (B). EAE-AS and anti-MOG showed high levels of binding on EL4-MOG cells (DAB staining), whereas NAIVE-AS did not bind (C). Magnification=40X, Scale=100Οm. (JPEG 344 kb