Irradiation of animals and transport to site of analysis
All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), after approval from the local ethical committees (Uppsala University and the Agricultural Research Council) and by the Swedish Committee for Ethical Experiments on Laboratory Animals. Male NMRI mice (Charles River, Germany) were exposed to a single dose of total body gamma irradiation of 0 (sham), 0.02, 0.1, 0.5 and 1.0 Gy on PND 10 (
60Co source, 0.025 Gy/min) (The Svedberg laboratory, Uppsala University) without anaesthesia. Neonates were not restrained during irradiation and could freely move during treatment. Dose verification was done with an ionisation chamber (Markus chamber type 23343, PTW-Freiburg). Three litters were used within each irradiation group to minimise inter-litter effects. Mice were kept at Uppsala University for behavioural testing until age of 5 months and were sent then to Germany receiving a routine treatment for intestinal parasites with Ivomac (Merial, 0.03 mg/mouse, over 1 week) [
55] and Italy. At age of 6 months (histological analysis) or 7 months (all other analyses), animals were sacrificed. Animals were kept at all times under normal housing conditions [
8]. Animals were sacrificed via cervical dislocation (for immunofluorescence and immunohistochemistry) and CO
2 (other experiments).
Mass spectrometry-based proteome analysis
Brains were removed to ice-cold phosphate-buffered saline (PBS), rinsed and stereomicroscopically dissected under cold conditions. Hippocampi and cortices without meninges from each hemisphere were separately collected, gently rinsed in ice-cold PBS and snap-frozen in liquid nitrogen. Samples were stored at -20°C for further analysis. Samples were processed for proteomic analysis as previously described [
58]. Protein extracts from hippocampi and cortices were labelled with isotope coded protein label (ICPL) triplex reagent (SERVA Electrophoresis GmbH, Germany) according to the manufacturer’s instructions. Individual protein lysates (50 μg in 20 μl of 6 M guanidine hydrochloride from each cortex and hippocampus sample) were reduced, alkylated and labelled with the respective ICPL-reagent as follows: sham-irradiated sample with ICPL-0, 0.02 Gy or 0.1 Gy treated sample with ICPL-4 and 0.5 Gy or 1.0 Gy treated samples with ICPL-6. The labelled samples from five cortices and four hippocampi from each irradiation group were combined (sham-0.02Gy-0.5Gy and sham-0.1Gy-1.0Gy) followed by overnight precipitation with 80% acetone at -20°C to purify the labelled protein content. Biological replicates included animals from three different litters. Precipitates were suspended in Laemmli buffer and separated by 12% SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining. Gel lanes were horizontally cut into six equal slices, destained and trypsinised overnight as described recently [
59]. Peptides were extracted and acidified with 1% formic acid and analysed via mass spectrometry.
LC-MS/MS analysis was performed on a LTQ-Orbitrap XL (Thermo Fisher, Germany) as described previously [
60]. Briefly, the gel slice fractionated samples were automatically injected and loaded onto the trap column and after 5 min peptides were eluted and separated on the analytical column by reversed phase chromatography operated on a nano-HPLC (Ultimate 3000, Dionex, Germany) with a nonlinear 170 min gradient using 35% acetonitrile in 0.1% formic acid in water (A) and 0.1% formic acid in 98% acetonitrile (B) at a flow rate of 300 nl/min. The gradient settings were: 5–140 min: 14.5-90% A, 140–145 min: 90% A - 95% B, 145–150 min: 95% B and equilibration for 15 min to starting conditions. From the MS pre-scan, the 10 most abundant peptide ions were selected for fragmentation in the linear ion trap if they exceeded an intensity of at least 200 counts and were at least doubly charged. During fragment analysis, a high-resolution (60,000 full-width half maximum) MS spectrum was acquired in the Orbitrap with a mass range from 200 to 1500 Da.
MS/MS spectra were searched against the ENSEMBL mouse database via MASCOT software with a mass error and fragment tolerance of 10 ppm and 0.6 Da, including not more than one missed cleavage. Fixed and variable modifications were carbamidomethylation of cysteine and ICPL-0, ICPL-4 and ICPL-6 for lysine. Proteins were identified and quantified based on the ICPL pairs using the Proteome Discoverer software (Version 1.3 – Thermo Fisher, Germany). To ensure that only high-confident peptides were used for protein quantification, we applied the MASCOT percolator algorithm [
61] as used previously [
62,
63]. The q value of the percolator algorithm was set to 0.01 representing strict peptide ranking. Thus, only the best ranked peptides were used. Further, these peptides were filtered against a Decoy database resulting in a false discovery rate (FDR) of each LC-MS-run; the significance threshold of the FDR was set to 0.01 to ensure that only highly confident peptides were used for protein quantification. Proteins from each LC-MS-run were normalised against the median of all quantifiable proteins. Proteins were considered significantly deregulated if they fulfilled the following criteria: (i) identified by at least two unique peptides in three out of the four hippocampal biological replicates and four out of the five cortical biological replicates, (ii) quantified with a ICPL-4/ICPL-0 and ICPL-6/ICPL-0 variability of ≤ 30% and (iii) had a fold-change of ≥ 1.3 or ≤ -1.3. The threshold of ±1.3 is based on the average experimental technical variance of the multiple analysis of hippocampal and cortical technical replicates (13.8%) (Additional file
1: Table S1). Further, it enables the unbiased confident quantification of deregulated proteins regardless of applied radiation dose (Additional file
1: Table S2).
Immunoblotting analysis
Hippocampal and cortical protein extracts (15 μg) were separated on 12% SDS polyacrylamide gels and were blotted to nitrocellulose membranes (GE Healthcare, Germany) via BIO-RAD criterion™ Blotter system at 100 V for 2 h. Membranes were blocked with Roti
R-Block solution (Roth, Germany), washed and incubated overnight at 4°C with primary antibody dilutions as indicated by the manufacturer (GAPDH – sc-47724 Santa Cruz, Germany; CDC42 – ab106374 Abcam, Germany; Rac1 – ab33186 Abcam, Germany; p-PAK1 – 2601 Cell Signaling, Germany; Fascin – ab74487 Abcam, Germany; Cofilin – 3312 Cell Signaling, Germany; Rho-GDIα – ab108977 Epitomics, Germany; p-LIMK1/2 – sc-28409-R Santa Cruz, Germany; p-Cofilin – 3311 Cell Signalling, Germany; LIMK1 – 3842 Cell Signaling, Germany; p-Rho-GDIα – sc-33047 Santa Cruz, Germany; TNFα – 3707 Cell Signaling, Germany; p-IGF1Rβ/INSRβ – 3021 Cell Signalling, Germany; Arc – ab118929 Abcam, Germany; c-Fos – sc-52 Santa Cruz, Germany; p-CREB – 9191 Cell Signaling, Germany; CREB – 4820 Cell Signaling, Germany). After a washing step, blots were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies in 8% milk for 1 h at room temperature and developed using the ECL system (GE Healthcare, Germany) using standard procedures. GAPDH was not significantly deregulated either at the mRNA or protein level in any sample and was therefore used as the loading control. Each irradiated group was run on separate immunoblots with corresponding sham-irradiated control samples under identical conditions on the same day. Immunoblots were only considered for quantification (TotalLab TL100 software;
http://www.totallab.com) if the ratios between control samples and endogenous GAPDH were similar after software-suggested background correction. Three biological replicates were used for statistical analysis (unpaired Student’s t-test) with a significance threshold of 0.05.
Detection of global lipid peroxidation was done via quantification of malondialdehyde-tagged proteins using a primary antibody against malondialdehyde (MDA11-S Alpha Diagnostic, USA). A total of four immunoblots (50 μg protein lysate per lane) were run on the same day under identical conditions using the corresponding three control samples on each blot and four biological samples from each irradiation group. Immunoblots were considered for quantification if (i) the pattern and intensity of lanes stained with Ponceau S were equal and (ii) total lane intensity of malondialdehyde-tagged proteins was similar. Five bands per lane in the range of 35 – 55 kDa were selected as representative indicators of global lipid peroxidation and were used for further quantification. Each band was normalised against the total lane intensity and fold-changes were calculated from each blot separately. Three and four biological replicates of control and irradiated samples were used for statistical analysis, respectively. Data analysis was done from two independent technical experiments (Student’s t-test, unpaired).
Transcriptomics and microRNA analysis
Total RNA from frozen hippocampi and cortices was isolated and purified using the mirVana™ Isolation Kit (Ambion, Germany) according to the manufacturer’s protocol. For both microRNA and gene expression studies the OD ratio of 260 nm / 280 nm from RNA lysates was estimated using a Nanodrop spectrophotometer. This ratio reflecting the RNA quality ranged between 2.0 and 2.1. Obtained RIN values ranged between 8.6 and 9.0 (TapeStation, Lab901). Eluates were stored at -20°C until further analysis.
Hippocampal and cortical RNA isolates (100 ng) were used to quantify the gene expression of 84 genes [“synaptic plasticity” (sham, 0.5 Gy, 1.0 Gy), “PI3k/Akt signalling pathway” (sham, 0.5 Gy, 1.0 Gy) and “circadian rhythm” (sham, 1.0 Gy) (RT
2 Profiler PCR array – Qiagen, Germany)]. The assays were performed following manufacturer’s instructions on a StepOnePlus (Applied Biosystems, Germany) using RT
2SYBR Green Mastermix. For pathway-focused transcriptomics, the relative expression of each mRNA was normalised against the median of all 84 target genes using the equation 2
-ΔΔCt, where ΔΔCt = ΔCt
irradiated – ΔCt
sham and ΔCt = Ct
target-mRNA – Ct
median-of-84-target-genes. Three biological replicates from three different litters were used within each dose group. Gene expression changes were considered significant if they reached a p-value of ≤ 0.05 and had a fold-change of ≥ 1.2 or ≤ -1.2. The threshold of ± 1.2 is based on the average experimental technical variance (8.4%) and biological variance (6.9%) of a set of 14 overlapping target genes (Additional file
1: Table S3). Results from overlapping gene targets were only regarded as significantly deregulated if they (i) were consistently up- or down-regulated, (ii) had overlapping confidence intervals and (ii) were consistently significantly changed.
Expression of single miRNAs using the QuantiTect Reverse Transcription Kit (Qiagen, Germany) and single miRNAs using the TaqMan Single MicroRNA Assay (Applied Biosystems, Germany) were performed following manufacturer’s protocol on a StepOnePlus device (Applied Biosystems, Germany) using Taqman-primers. For single mRNA and miRNA quantification, following Taqman-primers were used: mmu-miR-132 (ID000457), mmu-miR-134 (ID001186), mmu-miR-212 (ID002551), snoRNA135 (ID001239),
Tnfα (Mm00443260_g1),
Gapdh (Mm99999915_g1) and
Limk1 (Mm01196310_m1) - all from Life Technologies, Germany. Potential contamination with genomic DNA was excluded using the same conditions but without reverse transcriptase. Expression levels of miRNA and mRNA were calculated based on the 2
-ΔΔCt method with normalisation against endogenous snoRNA135 [
64] and
Gapdh, respectively. Changes were considered significant if they reached a p-value of ≤ 0.05 (unpaired Student’s t-test, n = 3).
Immunohistochemistry and immunofluorescence
Formalin fixed and paraffin embedded tissues were prepared using standard techniques [
65]. For immunohistochemistry, one μm thick single (stainings of NeuN and Ki67) and 4 μm thick serial (stainings of Caspase-3, GFAP, Sox2, Dcx, CD11b, PCNA and γH2AX) sagittal whole brain sections were dewaxed, rehydrated and heated in citrate buffer for 30 minutes. Quenching of endogenous peroxidase was performed with 3% H
2O
2 in methanol. Brain sections were incubated with primary antibody dilutions as indicated by the manufacturer. Following antibodies were used for immunohistochemistry: Caspase-3 (9664 – Cell Signaling, Germany), GFAP (Z0334 – Dako, Germany), Sox2 (ab97959 – Abcam, Germany), Dcx (ab97959 – Abcam, Germany), CD11b (ab1211 – Abcam, Germany), PCNA (NA03 mAB-1 – Calbiochem, Germany), γH2AX (05636 – Upstate Biotechnologies Inc., USA), Ki67 (ab15580 – Abcam, Germany) and NeuN (MAB377 – Millipore, Germany). Immunohistochemical analysis was done using polyclonal antibodies against Sox2, Dcx and CD11b. Complexes were visualised using a rabbit biotinylated-conjugated secondary antibody; after incubation with avidin-biotin immunoperoxidase staining, the antibody-antigen complexes were visualised with Vector NovaRED Substrate Kit (Vector Laboratories Inc., CA, USA) according to manufacturer’s instructions. Antibody–antigen complexes of polyclonal GFAP or caspase-3 were visualised using horseradish peroxidase-conjugated secondary antibody and the DAB chromogen system (Dako North American Inc, CA, USA). Immunohistochemical analysis of monoclonal antibody against γ-H2AX and PCNA was performed using the HistoMouse MAX Kit (Invitrogen Corporation, CA). The staining against NeuN was perfomed using the MoMap Kit (760–137 - Ventana, Germany), according to manufacturer’s instructions. Antibody-antigen complexes of Ki67 and NeuN were visualised using soluble immune complex of biotinylated secondary and mouse primary antibody (MoMap Kit 760–137 - Ventana, Germany) or rabbit biotinylated-conjugated secondary antibody. Subsequently, slides were incubated with avidin-biotin horseradish immunoperoxidase and were visualised with diaminobenzidine (DAB) (Sigma Aldrich, Germany).
The number of positive cells for GFAP or Sox2 in the subgranular zone (SGZ) was determined and expressed as a fraction of labelled cells out of the total number of granule neurons in the DG. To quantify PCNA or Dcx, the number of positive cells in the SGZ was expressed per μm2 of DG. Immunohistochemical staining for NeuN was performed to assess the neuronal density in the granule cell layer of the DG. Counting was carried out in a rectangular field of 4000 μm2 in the supra- and infrapyramidal blade and in the crest area of the DG. The number of positive cells in each of the areas was recorded separately followed by statistical analysis of the mean from three biological replicates. For quantification of microglia in brain sections immunostaining for CD11b was imaged by HistoFAXS (TissueGnostic, Austria). Regions of interest were selected in the molecular layer, DG and hilus and analysed with HistoQuest (TissueGnostics, Austria) using automatic colour separation and quantification. Quantitative analysis of astroglial cells (labelled by GFAP antibody) was performed by counting positive cells in the hippocampus (H) area (GFAP cells/H area [μm2]). Both DG and H area were measured by imaging software NIS-Elements BR4.00.05 (Nikon, Instruments Europe B.V., I) after tracing the DG / H outline.
All images were analysed using identical software settings. Statistical analysis (Student’s t-test, unpaired) was performed with six biological replicates for stainings of Caspase-3, GFAP, Sox2, Dcx, CD11b, PCNA and γH2AX and three biological replicates for stainings of Ki67 and at least three biological replicates for NeuN. Differences were considered to be significant when p-values were < 0.05 using unpaired Student’s t-test.
For immunofluorescence, one μm thick brain sagittal sections were dewaxed, rehydrated and heated in citrate buffer followed by auto-fluorescence block (0.1% sudan black in 70% ethanol). After a goat serum block, slides were overnight incubated with rabbit anti-mouse primary antibody against MAP-2 (ab32454 – Abcam, Germany) followed by goat anti-rabbit Cy3-Fab-fragment IgG secondary antibody (111-167-003 - Jackson ImmunoResearch, UK) after manufacturer’s instructions. Subsequently, the slides were washed in PBS and overnight incubated with rabbit anti-mouse primary antibody against PSD-95 (ab18258 – Abcam, Germany) followed by goat anti-rabbit Alexa-fluor IgG secondary antibody (111-545-144 - Jackson ImmunoResearch, UK) after manufacturer’s indications. Subsequently, the slides were nuclear stained with Hoechst and mounted with antifade fluorescence mounting media. Sample processing was done under identical conditions on the same day. All images were analysed using identical software settings. The MAP-2 / PSD-95 intensity in the region of interest was normalised against the Hoechst intensity within this region. Three biological replicates were used in all cases. Statistical significance was calculated with unpaired Student’s t-test.